Method of inducing an immune response by administering WDRPUH epitope peptides

ABSTRACT

The present invention provides peptides containing the amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 16, 17, 30, 31, 34, 36, 37, 40, 41, 45, 49, 55, 57 and 61, as well as peptides containing the above-mentioned amino acid sequences in which 1, 2, or several amino acid(s) are substituted, deleted, inserted or added, but still have cytotoxic T cell inducibility. The present invention also provides drugs for treating or preventing tumors, which drugs containing these peptides. The peptides of the present invention can also be used as vaccines.

PRIORITY

The present application is a division of U.S. application Ser. No.13/900,720, filed May 23, 2013, which is a division of U.S. applicationSer. No. 13/132,891, filed Aug. 17, 2011, now U.S. Pat. No. 8,541,546,which is a U.S. National Stage Application of PCT/JP2009/006573, filedDec. 3, 2009, which claims the benefit of U.S. Provisional ApplicationsNo. 61/200,962, filed on Dec. 5, 2008, and 61/209,704, filed on Mar. 9,2009, the entire contents of which are incorporated by reference hereinin their entirety.

REFERENCE TO SEQUENCE LISTING

This application includes a Sequence Listing as a text file named“87331-949057-SEQLIST.txt” created Jul. 8, 2015, and containing 28,948bytes. The material contained in this text file is incorporated byreference in its entirety for all purposes.

TECHNICAL FIELD

The present invention relates to the field of biological science, morespecifically to the field of cancer therapy. In particular, the presentinvention relates to novel peptides that are extremely effective ascancer vaccines, and drugs for treating and preventing tumors.

BACKGROUND ART

It has been demonstrated that CD8 positive cytotoxic T lymphocytes(CTLs) recognize epitope peptides derived from the tumor-associatedantigens (TAAs) found on major histocompatibility complex (MHC) class Imolecules, and then kill the tumor cells. Since the discovery of themelanoma antigen (MAGE) family as the first example of TAAs, many otherTAAs have been discovered, primarily through immunological approaches(Boon T, Int J Cancer 1993 May 8, 54(2): 177-80; Boon T & van derBruggen P, J Exp Med 1996 Mar. 1, 183(3): 725-9). Some of these TAAs arecurrently undergoing clinical development as immunotherapeutic targets.

Identification of new TAAs, capable of inducing potent and specificanti-tumor immune responses, warrants further development and clinicalapplication of peptide vaccination strategies for various types ofcancer (Harris C C, J Natl Cancer Inst 1996 Oct. 16, 88(20): 1442-55;Butterfield L H et al., Cancer Res 1999 Jul. 1, 59(13): 3134-42; VissersJ L et al., Cancer Res 1999 Nov. 1, 59(21): 5554-9; van der Burg S H etal., J Immunol 1996 May 1, 156(9): 3308-14; Tanaka F et al., Cancer Res1997 Oct. 15, 57(20): 4465-8; Fujie T et al., Int J Cancer 1999 Jan. 18,80(2): 169-72; Kikuchi M et al., Int J Cancer 1999 May 5, 81(3): 459-66;Oiso M et al., Int J Cancer 1999 May 5, 81(3): 387-94). To date, therehave been several reports of clinical trials using thesetumor-associated antigen derived peptides. Unfortunately, only a lowobjective response rate has been observed in these cancer vaccine trialsso far (Belli F et al., J Clin Oncol 2002 Oct. 15, 20(20): 4169-80;Coulie P G et al., Immunol Rev 2002 October, 188: 33-42; Rosenberg S Aet al., Nat Med 2004 September, 10(9): 909-15).

As a target for immunotherapy, TAAs indispensable for the proliferationand survival of cancer cells are suited, because the use of such TAAsmay minimize the well-described risk of immune escape of cancer cellsattributable to deletion, mutation, or down-regulation of TAAs as aconsequence of therapeutically driven immune selection.

WDRPUH was identified as a novel WD repeat protein that is upregulatedin hepatocellular carcinoma through gene expression profile using agenome-wide cDNA microarray containing 23,040 genes (Silva et al.,Neoplasia 2005 April; 7(4):348-55, WO 2003/104276). WD repeat-containingproteins have been reported to play crucial roles in a wide range ofphysiologic functions, including signal transduction, RNA processing(Bjorn et al., Mol Cell Biol. 1989 September; 9(9):3698-709.),remodeling of the cytoskeleton (Vaisman et al., Mol Gen Genet. 1995 Apr.20; 247(2):123-36), regulation of vesicular traffic (Pryer et al., JCell Biol. 1993 February; 120(4):865-75), and cell division (Feldman etal., Cell. 1997 Oct. 17; 91(2):221-30). Northern blot analysisdemonstrated that WDRPUH was over-expressed at a significantly highlevel in a great majority of hepatocellular carcinoma, but was notexpressed in normal organs except for testis. Furthermore, suppressionof WDRPUH expression by siRNA was shown to significantly inhibit growthof human hepatocellular carcinoma cell lines (Silva et al., Neoplasia2005 April; 7(4):348-55, WO 2003/104276).

CITATION LIST Patent Literature

-   [PTL 1] WO 2003/104276

Non Patent Literature

-   [NPL 1] Boon T, Int J Cancer 1993 May 8, 54(2): 177-80-   [NPL 2] Boon T & van der Bruggen P, J Exp Med 1996 Mar. 1, 183(3):    725-9-   [NPL 3] Harris C C, J Natl Cancer Inst 1996 Oct. 16, 88(20) 1442-55-   [NPL 4] Butterfield L H et al., Cancer Res 1999 Jul. 1, 59(13),    3134-42-   [NPL 5] Vissers J L et al., Cancer Res 1999 Nov. 1, 59(21): 5554-9-   [NPL 6] van der Burg S H et al., J Immunol 1996 May 1, 156(9):    3308-14-   [NPL 7] Tanaka F et al., Cancer Res 1997 Oct. 15, 57(20): 4465-8-   [NPL 8] Fujie T et al., Int J Cancer 1999 Jan. 18, 80(2): 169-72-   [NPL 9] Kikuchi M et al., Int J Cancer 1999 May 5, 81(3): 459-66-   [NPL 10] Oiso M et al., Int J Cancer 1999 May 5, 81(3): 387-94-   [NPL 11] Belli F et al., J Clin Oncol 2002 Oct. 15, 20(20): 4169-80-   [NPL 12] Coulie P G et al., Immunol Rev 2002 October, 188: 33-42-   [NPL 13] Rosenberg S A et al., Nat Med 2004 September, 10(9): 909-15-   [NPL 14] Silva et al., Neoplasia 2005 April; 7(4):348-55-   [NPL 15] Bjorn et al., Mol Cell Biol. 1989 September; 9(9):3698-709-   [NPL 16] Vaisman et al., Mol Gen Genet. 1995 Apr. 20; 247(2):123-36)-   [NPL 17] Pryer et al., J Cell Biol. 1993 February; 120(4):865-75-   [NPL 18] Feldman et al., Cell. 1997 Oct. 17; 91(2):221-30

SUMMARY OF INVENTION

The present invention is based in part on the discovery of suitabletargets of immunotherapy. Because TAAs are generally perceived by theimmune system as “self” and therefore often have no innateimmunogenicity, the discovery of appropriate targets is of extremeimportance. As noted above, recognizing that WDRPUH (SEQ ID NO: 64encoded by the gene of GenBank Accession No. NM_145054 (SEQ ID NO: 63))has been identified as up-regulated in cancer tissue of hepatocellularcarcinoma, WDRPUH is a candidate target for immunotherapy.

The present invention is based, at least in part, on the identificationof specific epitope peptides of the gene products of WDRPUH whichpossess the ability to induce CTLs specific to WDRPUH. As discussed indetail below, peripheral blood mononuclear cells (PBMCs) obtained from ahealthy donor were stimulated using HLA-A*2402 or HLA-A*0201 bindingcandidate peptides derived from WDRPUH. CTL lines with specificcytotoxicity against HLA-A24 or HLA-A2 positive target cells pulsed witheach of candidate peptides were then established. The resultsdemonstrated that the peptides are HLA-A24 or HLA-A2 restricted epitopepeptides that can induce potent and specific immune responses againstcells expressing WDRPUH on the surface. Further, it indicated thatWDRPUH is strongly immunogenic and the epitopes thereof are effectivetargets for tumor immunotherapy.

Accordingly, it is an object of the present invention to provideisolated peptides that bind to the HLA antigen, which peptides consistof WDRPUH (SEQ ID NO: 64) or a fragment of WDRPUH. Such peptides areexpected to have CTL inducibility and can be used for inducing CTLs inex vivo or for administration to a subject for inducing immune responsesagainst cancers such as hepatocellular carcinoma. The peptides may benonapeptides or decapeptides preferably consisting of the amino acidsequence selected from among SEQ ID NOs: 1, 2, 3, 4, 16, 17, 30, 31, 34,36, 37, 40, 41, 45, 49, 55, 57 and 61, which show strong CTLinducibility.

In addition, the present invention contemplates modified peptides,having an amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 16, 17, 30, 31,34, 36, 37, 40, 41, 45, 49, 55, 57 and 61 wherein one, two or more aminoacids are substituted, inserted, deleted or added, so long as themodified peptides retain the original CTL inducibility.

It is a further object of the present invention to provide isolatedpolynucleotides encoding any of the peptides of the present invention.These polynucleotides can be used for inducing antigen-expressing cells(APCs) with CTL inducibility or for administration to a subject toinduce immune responses against the present peptides and thus finallyagainst cancers.

When administered to a subject, the present peptides are presented onthe surface of APCs and then induce CTLs targeting the respectivepeptides. Therefore, it is an object of the present invention to provideagents containing any of the peptides or polynucleotides of the presentinvention for inducing CTLs. These agents containing any of the peptidesor polynucleotides of the present invention can be used for thetreatment and/or prophylaxis of cancers, such as hepatocellularcarcinoma, and/or the prevention of postoperative recurrence thereof.Thus, it is yet another object of the present invention to providepharmaceutical agents for the treatment and/or prophylaxis of cancers,and/or prevention of postoperative recurrence thereof, which containsany of the peptides or polynucleotides of the present invention. Thepresent agents or pharmaceutical agents may also contain, as the activeingredient, APCs or exosomes which present any of the present peptidesinstead of or in addition to the present peptides or polynucleotides.

The peptides or polynucleotides of the present invention have theability to induce APCs which present, on its surface, a complex of anHLA antigen and the present peptide. For example, the induction can beachieved by contacting APCs derived from a subject with a peptide of thepresent invention or introducing a polynucleotide encoding a peptide ofthe present invention into APCs. Such APCs have high CTL inducibilityagainst the target peptides and are useful for cancer immunotherapy.Therefore, it is a further object of the present invention to providemethods for inducing APCs with CTL inducibility and APCs obtained by themethods.

It is yet another object of the present invention to provide methods forinducing CTLs, which methods contain the step of co-culturingCD8-positive cells with APCs or exosomes presenting a peptide of thepresent invention on its surface or the step of introducing apolynucleotide into a T cell, which polynucleotide encodes a T cellreceptor (TCR) subunit polypeptide binding to a peptide of the presentinvention. The CTLs obtained by the methods are useful for treatingand/or preventing cancers, such as hepatocellular carcinoma. Therefore,it is a further object of the present invention to provide CTLs obtainedby any of the present methods.

Another object of the present invention to provide methods for inducingimmune response against cancers, which methods contain the step ofadministering an agent containing any of the WDRPUH polypeptides,polynucleotides encoding WDRPUH polypeptides, exosomes or the APCspresenting WDRPUH polypeptides of the present invention.

The present invention find use for application to any diseases relatedto WDRPUH over-expression including, but are not limited to, cancer,particularly hepatocellular carcinoma.

It is to be understood that both the foregoing summary of the inventionand the following detailed description are of exemplified embodiments,and not restrictive of the invention or other alternate embodiments ofthe invention.

BRIEF DESCRIPTION OF DRAWINGS

Various aspects and applications of the present invention will becomeapparent to the skilled artisan upon consideration of the briefdescription of the figures and the detailed description of the presentinvention and its preferred embodiments which follows.

FIGS. 1a-1f include a series of photographs depicting the results ofIFN-gamma ELISPOT assay on CTLs that were induced with peptides derivedfrom WDRPUH. The CTLs in well numbers #3 and #6 stimulated withWDRPUH-A24-9-40 (SEQ ID NO: 1) (a), #8 with WDRPUH-A24-9-314 (SEQ ID NO:2) (b), #2 and #6 with WDRPUH-A24-9-509 (SEQ ID NO: 3) (c), #1, #2 and#5 with WDRPUH-A24-9-339 (SEQ ID NO: 4) (d), #2, #3, #4, #6, #7 and #8with WDRPUH-A24-10-409 (SEQ ID NO: 16) (e) and #5, #6 and #8 withWDRPUH-A24-10-40 (SEQ ID NO: 17) (f) showed potent IFN-gamma productionas compared with control, respectively. The cells in the wells denotedwith a rectangular box were expanded to establish CTL lines. In thefigure, “+” indicates that the cells in the wells were pulsed withappropriate peptides, and “−” indicates that the cells had not beenpulsed with any peptides.

FIGS. 2a-2f include a series of line graphs depicting IFN-gammaproduction of CTL lines stimulated with WDRPUH-A24-9-40 (SEQ ID NO: 1)(a), WDRPUH-A24-9-314 (SEQ ID NO: 2) (b), WDRPUH-A24-9-509 (SEQ ID NO:3) (c), WDRPUH-A24-9-339 (SEQ ID NO: 4) (d), WDRPUH-A24-10-409 (SEQ IDNO: 16) (e) and WDRPUH-A24-10-40 (SEQ ID NO: 17) (f) with IFN-gammaELISA assay. CTL lines established by stimulation with each of thepeptides showed potent IFN-gamma production as compared with control. Inthe figure, “+” indicates that the cells in the wells were pulsed withappropriate peptides, and “−” indicates that the cells had not beenpulsed with any peptides.

FIG. 3 is composed of a line graph depicting specific CTL activityagainst target cells that exogenously express WDRPUH and HLA-A*2402. COS7 cells transfected with only HLA-A*2402 or with only the full lengthWDRPUH gene were prepared as control. The CTL line established withWDRPUH-A24-9-314 (SEQ ID NO: 2) showed specific CTL activity against COS7 cells transfected with both WDRPUH and HLA-A*2402 (black lozenge). Incontrast, no significant specific CTL activity was detected againsttarget cells expressing either HLA-A*2402 (triangle) or WDRPUH (circle).

FIGS. 4a-4h include a series of photographs depicting the results ofIFN-gamma ELISPOT assay on CTLs that were induced with peptides derivedfrom WDRPUH. The CTLs in well numbers #2 and #7 stimulated withWDRPUH-A2-9-39 (SEQ ID NO: 30) (a), #2 with WDRPUH-A2-9-407 (SEQ ID NO:31) (b), #3 with WDRPUH-A2-9-288 (SEQ ID NO: 34) (c), #6 withWDRPUH-A2-9-237 (SEQ ID NO: 36) (d), #4 with WDRPUH-A2-9-543 (SEQ ID NO:37) (e), #4 with WDRPUH-A2-10-570 (SEQ ID NO: 40) (f), #2 and #8 withWDRPUH-A2-10-263 (SEQ ID NO: 41) (g), #5 with WDRPUH-A2-10-78 (SEQ IDNO: 45) (h) showed potent IFN-gamma production as compared with thecontrol, respectively. The cells in the wells denoted with a rectangularbox were expanded to establish CTL lines. In the figure, “+” indicatesthat the cells in the wells were pulsed with appropriate peptides, and“−” indicates that the cells had not been pulsed with any peptides.

FIGS. 4i-4l include a series of photographs depicting the results ofIFN-gamma ELISPOT assay on CTLs that were induced with peptides derivedfrom WDRPUH. The CTLs in well numbers #2 stimulated with WDRPUH-A2-10-10(SEQ ID NO: 49) (i), #6 with WDRPUH-A2-10-411 (SEQ ID NO: 55) (j), #7with WDRPUH-A2-10-287 (SEQ ID NO: 57) (k) and #6 with WDRPUH-A2-10-265(SEQ ID NO: 61) (1) showed potent IFN-gamma production as compared withthe control, respectively. The cells in the wells denoted with arectangular box were expanded to establish CTL lines. In the figure, “+”indicates that the cells in the wells were pulsed with appropriatepeptides, and “−” indicates that the cells had not been pulsed with anypeptides.

FIGS. 5a-5b are composed of line graphs depicting the IFN-gammaproduction of CTL lines stimulated with SEQ ID NO: 30 (a) and SEQ ID NO:34 (b) detected by IFN-gamma ELISA assay. CTL lines established bystimulation with each peptide showed potent IFN-gamma production ascompared with the control. In the figure, “+” indicates that the cellsin the wells were pulsed with appropriate peptides, and “−” indicatesthat the cells had not been pulsed with any peptides. FIGS. 5c-5d depictthe IFN-gamma production of the CTL clones established by limitingdilution from the CTL lines stimulated with SEQ ID NO: 30 (c) and SEQ IDNO: 34 (d). The results depicted herein demonstrate that the CTL clonesestablished by stimulation with SEQ ID NO: 30 (c) and SEQ ID NO: 34 (d)showed potent IFN-gamma production as compared with the control. In thefigure, “+” indicates that the cells in the wells were pulsed with SEQID NO: 30 (c) and SEQ ID NO: 34 (d) and “−” indicates that the cells hadnot been with any peptides. FIG. 5e is composed of a line graphdepicting specific CTL activity against the target cells thatexogenously express WDRPUH and HLA-A*0201. COS 7 cells transfected withonly HLA-A*0201 or with only the full length WDRPUH gene were preparedas controls. The CTL clone established with WDRPUH-A2-9-288 (SEQ ID NO:34) showed specific CTL activity against COS 7 cells transfected withboth WDRPUH and HLA-A*0201 (black lozenge). In contrast, no significantspecific CTL activity was detected against target cells expressingeither HLA-A*0201 (triangle) or WDRPUH (circle).

DESCRIPTION OF EMBODIMENTS

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsof the present invention, the preferred methods, devices, and materialsare now described. However, before the present materials and methods aredescribed, it is to be understood that the present invention is notlimited to the particular sizes, shapes, dimensions, materials,methodologies, protocols, etc. described herein, as these may vary inaccordance with routine experimentation and optimization. It is also tobe understood that the terminology used in the descriptions is for thepurpose of describing the particular versions or embodiments only, andis not intended to limit the scope of the present invention which willbe limited only by the appended claims.

The disclosure of each publication, patent or patent applicationmentioned in this specification is specifically incorporated byreference herein in its entirety. However, nothing herein is to beconstrued as an admission that the invention is not entitled to antedatesuch disclosure by virtue of prior invention.

In case of conflict, the present specification, including definitions,will control. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

I. Definitions

The words “a”, “an”, and “the” as used herein mean “at least one” unlessotherwise specifically indicated.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is a modified residue, or a non-naturally occurring residue,such as an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

The term “amino acid” as used herein refers to naturally occurring andsynthetic amino acids, as well as amino acid analogs and amino acidmimetics that similarly function to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those modified after translation in cells (e.g.,hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine). The phrase“amino acid analog” refers to compounds that have the same basicchemical structure (an alpha carbon bound to a hydrogen, a carboxygroup, an amino group, and an R group) as a naturally occurring aminoacid but have a modified R group or modified backbones (e.g.,homoserine, norleucine, methionine, sulfoxide, methionine methylsulfonium). The phrase “amino acid mimetic” refers to chemical compoundsthat have different structures but similar functions to general aminoacids.

Amino acids may be referred to herein by their commonly known threeletter symbols or the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission.

The terms “polynucleotides”, “genes”, “nucleotides” and “nucleic acids”are used interchangeably herein unless otherwise specifically indicated.

Unless otherwise defined, the term “cancer” refers to the cancersover-expressing the WDRPUH gene, examples of which include, but are notlimited to, hepatocellular carcinoma.

Unless otherwise defined, the terms “cytotoxic T lymphocyte”, “cytotoxicT cell” and “CTL” are used interchangeably herein and, unless otherwisespecifically indicated, refer to a sub-group of T lymphocytes that arecapable of recognizing non-self cells (e.g., tumor cells, virus-infectedcells) and inducing the death of such cells.

Unless otherwise defined, the term “HLA-A2” contains the subtypes suchas HLA-A0201 or HLA-A0206.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

II. Peptides

To demonstrate that peptides derived from WDRPUH function as an antigenrecognized by CTLs, peptides derived from WDRPUH (SEQ ID NO: 64) wereanalyzed to determine whether they were antigen epitopes restricted byHLA-A24 which are commonly encountered HLA alleles (Date Y et al.,Tissue Antigens 47: 93-101, 1996; Kondo A et al., J Immunol 155:4307-12, 1995; Kubo R T et al., J Immunol 152: 3913-24, 1994).Candidates of HLA-A24 binding peptides derived from WDRPUH wereidentified based on their binding affinities to HLA-A24. The followingpeptides are the candidate peptides:

(SEQ ID NO: 1) WDRPUH-A24-9-40, (SEQ ID NO: 2) WDRPUH-A24-9-314,(SEQ ID NO: 3) WDRPUH-A24-9-509, (SEQ ID NO: 4) WDRPUH-A24-9-339,(SEQ ID NO: 16) WDRPUH-A24-10-409, and (SEQ ID NO: 17) WDRPUH-A24-10-40.

Candidates of HLA-A02 binding peptides derived from WDRPUH wereidentified based on their binding affinities to HLA-A02. The followingpeptides are the candidate peptides:

(SEQ ID NO: 30) WDRPUH-A2-9-39, (SEQ ID NO: 31) WDRPUH-A2-9-407,(SEQ ID NO: 34) WDRPUH-A2-9-288, (SEQ ID NO: 36) WDRPUH-A2-9-237,(SEQ ID NO: 37) WDRPUH-A2-9-543, (SEQ ID NO: 40) WDRPUH-A2-10-570,(SEQ ID NO: 41) WDRPUH-A2-10-263, (SEQ ID NO: 45) WDRPUH-A2-10-78,(SEQ ID NO: 49) WDRPUH-A2-10-10, (SEQ ID NO: 55) WDRPUH-A2-10-411,(SEQ ID NO: 57) WDRPUH-A2-10-287, and (SEQ ID NO: 61) WDRPUH-A2-10-265.

These established CTLs show potent specific CTL activity against targetcells pulsed with respective peptides. These results herein demonstratethat WDRPUH is an antigen recognized by CTLs and that the peptides maybe epitope peptides of WDRPUH restricted by HLA-A24 or HLA-A2.

Since the WDRPUH gene is over expressed in cancer cells of such ashepatocellular carcinoma and not in most normal organs, it is a goodtarget for immunotherapy. Thus, the present invention providesnonapeptides (peptides consisting of nine amino acid residues) anddecapeptides (peptides consisting of ten amino acid residues)corresponding to CTL-recognized epitopes of WDRPUH. Particularlypreferred examples of the peptides of the present invention includethose peptides consisting of the amino acid sequence selected from amongSEQ ID Nos: 1, 2, 3, 4, 16, 17, 30, 31, 34, 36, 37, 40, 41, 45, 49, 55,57 and 61.

Generally, software programs presently available on the Internet, suchas those described in Parker K C et al., J Immunol 1994 Jan. 1, 152(1):163-75, can be used to calculate the binding affinities between variouspeptides and HLA antigens in silico. Binding affinity with HLA antigenscan be measured as described, for example, in the reference to Parker KC et al., J Immunol 1994 Jan. 1, 152(1): 163-75; and Kuzushima K et al.,Blood 2001, 98(6): 1872-81. The methods for determining binding affinityare described, for example, in: Journal of Immunological Methods, 1995,185: 181-190; Protein Science, 2000, 9: 1838-1846. Therefore, one canselect fragments of WDRPUH, which have high binding affinity with HLAantigens using such software programs. Thus, the present inventionencompasses peptides consisting of any fragments derived from WDRPUH,which bind with HLA antigens identified using such known programs.Furthermore, the present peptide may also consist of the full length ofWDRPUH.

The peptides of the present invention can be flanked with additionalamino acid residues so long as the resulting peptide retains its CTLinducibility. The particular amino acid residues flanking the peptidesof the present invention can be composed of any kind of amino acids solong as they do not impair the CTL inducibility of the original peptide.Thus, the present invention also provides peptides having bindingability to HLA antigens and containing amino acid sequences derived fromWDRPUH. Such peptides are typically less than about 40 amino acids,often less than about 20 amino acids, usually less than about 15 aminoacids.

In general, the modification of one, two or more amino acids in apeptide will not influence the function of the peptide, and in somecases will even enhance the desired function of the original protein. Infact, modified peptides (i.e., peptides composed of an amino acidsequence in which one, two or several amino acid residues have beenmodified (i.e., substituted, deleted, added or inserted as compared toan original reference sequence) have been known to retain the biologicalactivity of the original peptide (Mark et al., Proc Natl Acad Sci USA1984, 81: 5662-6; Zoller and Smith, Nucleic Acids Res 1982, 10:6487-500; Dalbadie-McFarland et al., Proc Natl Acad Sci USA 1982, 79:6409-13). Thus, in one embodiment, the peptides of the present inventionmay have both CTL inducibility and an amino acid sequence selected fromamong SEQ ID NO: 1, 2, 3, 4, 16, 17, 30, 31, 34, 36, 37, 40, 41, 45, 49,55, 57 and 61, wherein one, two or even more amino acids are added,inserted, deleted and/or substituted.

Those skills in the art recognize that individual substitutions to anamino acid sequence which alters a single amino acid or a smallpercentage of amino acids tend to result in the conservation of theproperties of the original amino acid side-chain. As such, they areoften referred to as “conservative substitutions” or “conservativemodifications”, wherein the alteration of a protein results in amodified protein having a function analogous to the original protein.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Examples of amino acid side chaincharacteristics that are desirable to conserve include, for example,hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic aminoacids (R, D, N, C, E, Q, G, H, K, S, T), and side chains having thefollowing functional groups or characteristics in common: an aliphaticside-chain (G, A, V, L, I, P); a hydroxyl group containing side-chain(S, T, Y); a sulfur atom containing side-chain (C, M); a carboxylic acidand amide containing side-chain (D, N, E, Q); a base containingside-chain (R, K, H); and an aromatic containing side-chain (H, F, Y,W). In addition, the following eight groups each contain amino acidsthat are accepted in the art as conservative substitutions for oneanother:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Aspargine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins 1984).

Such conservatively modified peptides are also considered to be peptidesof the present invention. However, peptides of the present invention arenot restricted thereto and can include non-conservative modifications,so long as the modified peptide retains the CTL inducibility of theoriginal peptide. Furthermore, modified peptides should not exclude CTLinducible peptides of polymorphic variants, interspecies homologues, andalleles of WDRPUH.

To retain the requisite CTL inducibility one can modify (insert, delete,add and/or substitute) a small number (for example, 1, 2 or several) ora small percentage of the amino acids. Herein, the term “several” means5 or fewer amino acids, for example, 4, 3 or fewer. The percentage ofamino acids to be modified is preferably 20% or less, more preferably15% or less, even more preferably 10% or less, or 1 to 5%.

Moreover, amino acid residues may be substituted, inserted, deletedand/or added in the peptides to yield a modified peptide having improvedbinding affinity. When used in the context of immunotherapy, peptides ofthe present invention should be presented on the surface of a cell orexosome, preferably as a complex with an HLA antigen. In addition topeptides that are naturally displayed, since the regularity of thesequences of peptides displayed by binding to HLA antigens is alreadyknown (J Immunol 1994, 152: 3913; Immunogenetics 1995, 41: 178; JImmunol 1994, 155: 4307), modifications based on such regularity can beintroduced into the immunogenic peptides of the invention. For example,it may be desirable to substitute the second amino acid from theN-terminus substituted with phenylalanine, tyrosine, methionine, ortryptophan, and/or the amino acid at the C-terminus with phenylalanine,leucine, isoleucine, tryptophan, or methionine in order to increase theHLA-A24 binding. Thus, peptides having the amino acid sequences selectedfrom the group consisting of SEQ ID NOs: 1, 2, 3, 4, 16 and 17 whereinthe second amino acid from the N-terminus of the amino acid sequence ofthe SEQ ID NOs is substituted with phenylalanine, tyrosine, methionine,or tryptophan, and peptides, and/or wherein the C-terminus of the aminoacid sequence of the SEQ ID NOs is substituted with phenylalanine,leucine, isoleucine, tryptophan, or methionine are encompassed by thepresent invention. On the other hand, peptides possessing high HLA-A2binding affinity have their second amino acid from the N-terminussubstituted with leucine or methionine, and the amino acid at theC-terminus is substituted with valine or leucine. Thus, peptides havingthe amino acid sequences of SEQ ID NOs: 30, 31, 34, 36, 37, 40, 41, 45,49, 55, 57 and 61 wherein the second amino acid from the N-terminus issubstituted with leucine or methionine, and/or wherein the C-terminus issubstituted with valine or leucine are encompassed by the presentinvention. Substitutions can be introduced not only at the terminalamino acids but also at the position of potential T cell receptor (TCR)recognition of peptides. Several studies have demonstrated that apeptide with amino acid substitutions can be equal to or better than theoriginal, for example CAP1, p53₍₂₆₄₋₂₇₂₎, Her-2/neu₍₃₆₉₋₃₇₇₎ or gp100₍₂₀₉₋₂₁₇₎ (Zaremba et al. Cancer Res. 57, 4570-4577, 1997, T. K.Hoffmann et al. J Immunol. (2002) Feb. 1; 168(3):1338-47. S. O. Dionneet al. Cancer Immunol immunother. (2003) 52: 199-206 and S. O. Dionne etal. Cancer Immunology, Immunotherapy (2004) 53, 307-314).

The present invention also contemplates the addition of one, two orseveral amino acids to the N and/or C-terminus of the describedpeptides. Such modified peptides having high HLA antigen bindingaffinity and retaining CTL inducibility are also included in the presentinvention.

However, when the peptide sequence is identical to a portion of theamino acid sequence of an endogenous or exogenous protein having adifferent function, side effects such as autoimmune disorders and/orallergic symptoms against specific substances may be induced. Therefore,it is preferable to first perform homology searches using availabledatabases to avoid situations in which the sequence of the peptidematches the amino acid sequence of another protein. When it becomesclear from the homology searches that there exists not even a peptidewith 1 or 2 amino acid differences as compared to the objective peptide,the objective peptide can be modified in order to increase its bindingaffinity with HLA antigens, and/or increase its CTL inducibility withoutany danger of such side effects.

Although peptides having high binding affinity to the HLA antigens asdescribed above are expected to be highly effective, the candidatepeptides, which are selected according to the presence of high bindingaffinity as an indicator, are further examined for the presence of CTLinducibility. Herein, the phrase “CTL inducibility” indicates theability of the peptide to induce CTLs when presented onantigen-presenting cells (APCs). Further, “CTL inducibility” includesthe ability of the peptide to induce CTL activation, CTL proliferation,promote CTL lysis of target cells, and to increase CTL IFN-gammaproduction.

Confirmation of CTL inducibility is accomplished by inducing APCscarrying human MHC antigens (for example, B-lymphocytes, macrophages,and dendritic cells (DCs)), or more specifically DCs derived from humanperipheral blood mononuclear leukocytes, and after stimulation with thepeptides, mixing with CD8-positive cells, and then measuring theIFN-gamma produced and released by CTLs against the target cells. As thereaction system, transgenic animals that have been produced to express ahuman HLA antigen (for example, those described in BenMohamed L,Krishnan R, Longmate J, Auge C, Low L, Primus J, Diamond D J, HumImmunol 2000 August, 61(8): 764-79, Related Articles, Books, LinkoutInduction of CTL response by a minimal epitope vaccine in HLA A*0201/DR1transgenic mice: dependence on HLA class II restricted T(H) response)can be used. For example, the target cells can be radio-labeled with⁵¹Cr and such, and cytotoxic activity can be calculated fromradioactivity released from the target cells. Alternatively, CTLinducibility can be assessed by measuring IFN-gamma produced andreleased by CTLs in the presence of APCs that carry immobilizedpeptides, and visualizing the inhibition zone on the media usinganti-IFN-gamma monoclonal antibodies.

As a result of examining the CTL inducibility of the peptides asdescribed above, the nonapeptides or decapeptides having an amino acidsequence selected from among SEQ ID NOs: 1, 2, 3, 4, 16, 17, 30, 31, 34,36, 37, 40, 41, 45, 49, 55, 57 and 61 were found to exhibit particularlyhigh CTL inducibility as well as high binding affinity to an HLAantigen. Thus, these peptides are exemplified as preferred embodimentsof the present invention.

Furthermore, the result of homology analysis showed that these peptidesdo not have significant homology with peptides derived from any otherknown human gene products. This means that the possibility of unknown orundesired immune responses arising due to the use of the presentpeptides in immunotherapy is low. Therefore, also from this aspect,these peptides are preferable for eliciting immunity in cancer patientsagainst WDRPUH. Thus, particularly preferred peptides of the presentinvention include those having the amino acid sequence selected fromamong SEQ ID NOs: 1, 2, 3, 4, 16, 17, 30, 31, 34, 36, 37, 40, 41, 45,49, 55, 57 and 61.

In addition the above-described modifications, the peptides of thepresent invention can also be linked to other peptides, so long as theresulting peptide retains the requisite CTL inducibility of the originalpeptide. Examples of suitable peptides include, but are not limited to:the peptides of the present invention or CTL inducible peptides derivedfrom other TAAs. Linkers to be placed between the peptides are wellknown in the art and include, but are not limited to, for example, AAY(P. M. Daftarian et al., J Trans Med 2007, 5:26), AAA, NKRK (R. P. M.Sutmuller et al., J Immunol. 2000, 165: 7308-7315) and K (S. Ota et al.,Can Res. 62, 1471-1476, K. S. Kawamura et al., J Immunol. 2002, 168:5709-5715).

Furthermore, the peptides of the present invention can also be linked toother substances, so long as the resulting peptide retains the requisiteCTL inducibility of the original peptide. Examples of suitablesubstances include, but are not limited to: peptides, lipids, sugar andsugar chains, acetyl groups, natural and synthetic polymers, etc,provided the modifications do not destroy the biological activity of theoriginal peptide. The peptides can contain modifications such asglycosylation, side chain oxidation, or phosphorylation, etc, providedthe modifications do not destroy the biological activity of the originalpeptide. These kinds of modifications can be performed to conferadditional functions (e.g., targeting function, and delivery function)or to stabilize the polypeptide. For example, to increase the in vivostability of a polypeptide, it is known in the art to introduce D-aminoacids, amino acid mimetics or unnatural amino acids; this concept canalso be adapted to the present polypeptides. The stability of apolypeptide can be assayed in a number of ways. For instance, peptidasesand various biological media, such as human plasma and serum, can beused to test stability (see, e.g., Verhoef et al., Eur J Drug MetabPharmacokin 1986, 11: 291-302).

As noted above, it is possible to screen or select peptides that aremodified by substitution, insertion, deletion and/or addition of one,two or several amino acid residues, but still having the same or higheractivity as compared to the original peptide. Thus, the presentinvention also provides a method for screening or selecting a modifiedpeptide having the same or higher activity as compared to the originalpeptide. For example, such method may be composed of the steps asfollows:

a: modifying at least one amino acid residue in a peptide of the presentinvention by substitution, deletion, insertion and/or addition;

b: determining the activity of the modified peptide; and

c: selecting the peptide which was determined to have the same or higheractivity as compared to the original peptide.

Herein, the activity to be determined in step b may be the MHC bindingactivity, APC or CTL inducibility, and/or cytotoxic activity.

Herein, the peptides of the present invention can also be described as“WDRPUH peptide(s)” or “WDRPUH polypeptide(s)”.

III. Preparation of WDRPUH Peptides

The peptides of the invention can be prepared using well knowntechniques. For example, the peptides can be prepared synthetically,using recombinant DNA technology or chemical synthesis. The peptides ofthe invention can be synthesized individually or as longer polypeptidescomposed of two or more peptides. The peptides can then be isolatedi.e., purified or isolated so as to be substantially free of othernaturally occurring host cell proteins and fragments thereof, or anyother chemical substances.

A peptide of the present invention can be obtained through chemicalsynthesis based on the selected amino acid sequence. Examples ofconventional peptide synthesis methods that can be adapted for thesynthesis include, but are not limited to:

(i) Peptide Synthesis, Interscience, New York, 1966;

(ii) The Proteins, Vol. 2, Academic Press, New York, 1976;

(iii) Peptide Synthesis (in Japanese), Maruzen Co., 1975;

(iv) Basics and Experiment of Peptide Synthesis (in Japanese), MaruzenCo., 1985;

(v) Development of Pharmaceuticals (second volume) (in Japanese), Vol.14 (peptide synthesis), Hirokawa, 1991;

(vi) WO99/67288; and

(vii) Barany G. & Merrifield R. B., Peptides Vol. 2, “Solid PhasePeptide Synthesis”, Academic Press, New York, 1980, 100-118.

Alternatively, the present peptides can be obtained adapting any knowngenetic engineering methods for producing peptides (e.g., Morrison J, JBacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods inEnzymology (eds. Wu et al.) 1983, 101: 347-62). For example, first, asuitable vector harboring a polynucleotide encoding the objectivepeptide in an expressible form (e.g., downstream of a regulatorysequence corresponding to a promoter sequence) is prepared andtransformed into a suitable host cell. The host cell is then cultured toproduce the peptide of interest. The peptide can also be produced invitro adapting an in vitro translation system.

IV. Polynucleotides

The present invention also provides a polynucleotide which encodes anyof the aforementioned peptides of the present invention. These includepolynucleotides derived from the natural occurring WDRPUH gene (GenBankAccession No. NM_145697 (SEQ ID NO: 34)) as well as those having aconservatively modified nucleotide sequence thereof. Herein, the phrase“conservatively modified nucleotide sequence” refers to sequences whichencode identical or essentially identical amino acid sequences. Due tothe degeneracy of the genetic code, a large number of functionallyidentical nucleic acids encode any given protein. For instance, thecodons GCA, GCC, GCG, and GCU all encode the amino acid alanine Thus, atevery position where an alanine is specified by a codon, the codon canbe altered to any of the corresponding codons described without alteringthe encoded polypeptide. Such nucleic acid variations are “silentvariations,” which are one species of conservatively modifiedvariations. Every nucleic acid sequence herein which encodes a peptidealso describes every possible silent variation of the nucleic acid. Oneof ordinary skill in the art will recognize that each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine, andTGG, which is ordinarily the only codon for tryptophan) can be modifiedto yield a functionally identical molecule. Accordingly, each silentvariation of a nucleic acid that encodes a peptide is implicitlydescribed in each disclosed sequence.

The polynucleotide of the present invention can be composed of DNA, RNA,and derivatives thereof. A DNA is suitably composed of bases such as A,T, C, and G, and T is replaced by U in an RNA.

The polynucleotide of the present invention can encode multiple peptidesof the present invention with or without intervening amino acidsequences in between. For example, the intervening amino acid sequencecan provide a cleavage site (e.g., enzyme recognition sequence) of thepolynucleotide or the translated peptides. Furthermore, thepolynucleotide can include any additional sequences to the codingsequence encoding the peptide of the present invention. For example, thepolynucleotide can be a recombinant polynucleotide that includesregulatory sequences required for the expression of the peptide or canbe an expression vector (plasmid) with marker genes and such. Ingeneral, such recombinant polynucleotides can be prepared by themanipulation of polynucleotides through conventional recombinanttechniques using, for example, polymerases and endonucleases.

Both recombinant and chemical synthesis techniques can be used toproduce the polynucleotides of the present invention. For example, apolynucleotide can be produced by insertion into an appropriate vector,which can be expressed when transfected into a competent cell.Alternatively, a polynucleotide can be amplified using PCR techniques orexpression in suitable hosts (see, e.g., Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York,1989). Alternatively, a polynucleotide can be synthesized using thesolid phase techniques, as described in Beaucage S L & Iyer R P,Tetrahedron 1992, 48: 2223-311; Matthes et al., EMBO J 1984, 3: 801-5.

V. Exosomes

The present invention further provides intracellular vesicles calledexosomes, which present complexes formed between the peptides of thisinvention and HLA antigens on their surface. Exosomes can be prepared,for example by using the methods detailed in Japanese Patent ApplicationKohyo Publications Nos. Hei 11-510507 and WO99/03499, and can beprepared using APCs obtained from patients who are subject to treatmentand/or prevention. The exosomes of this invention can be inoculated asvaccines, in a fashion similar to the peptides of this invention.

The type of HLA antigens contained in the complexes must match that ofthe subject requiring treatment and/or prevention. For example, in theJapanese population, HLA-A24 and HLA-A2, particularly HLA-A2402 andHLA-A0201 or HLA-A0206 is prevalent and therefore would be appropriatefor treatment of Japanese patient. The use of the A24 type or the A2type that is highly expressed among the Japanese and Caucasian isfavorable for obtaining effective results, and subtypes such as A2402,A0201 or A0206 also find use. Typically, in the clinic, the type of HLAantigen of the patient requiring treatment is investigated in advance,which enables the appropriate selection of peptides having high levelsof binding affinity to the particular antigen, or having CTLinducibility by antigen presentation. Furthermore, in order to obtainpeptides having both high binding affinity and CTL inducibility,substitution, insertion, deletion and/or addition of 1, 2, or severalamino acids can be performed based on the amino acid sequence of thenaturally occurring WDRPUH partial peptide.

When using the A24 type HLA antigen for the exosome of the presentinvention, the peptides having the sequences selected from among SEQ IDNOs: 1, 2, 3, 4, 16, and 17 find use. Alternatively, when using the A2type HLA antigen for the exosome of the present invention, the peptideshaving a sequence of any one of SEQ ID NOs: 30, 31, 34, 36, 37, 40, 41,45, 49, 55, 57 and 61 find use.

VI. Antigen-Presenting Cells (APCs)

The present invention also provides isolated APCs that present complexesformed between HLA antigens and the peptides of this invention on itssurface. The APCs can be derived from patients who are subject totreatment and/or prevention, and can be administered as vaccines bythemselves or in combination with other drugs including the peptides ofthis invention, exosomes, or CTLs.

The APCs are not limited to a particular kind of cells and include DCs,Langerhans cells, macrophages, B cells, and activated T cells, which areknown to present proteinaceous antigens on their cell surface so as tobe recognized by lymphocytes. Since DC is a representative APC havingthe strongest CTL inducing action among APCs, DCs find use as the APCsof the present invention.

For example, an APC of the present invention can be obtained by inducingDCs from peripheral blood monocytes and then contacting (stimulating)them with the peptides of this invention in vitro, ex vivo or in vivo.The phrase “inducing APCs” includes contacting (stimulating) a cell withthe peptides of this invention, or nucleotides encoding the peptides ofthis invention to present complexes formed between HLA antigens and thepeptides of this invention on the cell's surface. When the peptides ofthis invention are administered to a subject, APCs that present thepeptides of this invention are induced in the body of the subject.Therefore, the APCs of this invention can be obtained by collecting theAPCs from a subject after administering a peptide of this invention tothe subject. Alternatively, the APCs of this invention can be obtainedby contacting APCs collected from a subject with the peptide of thisinvention.

The APCs of the present invention can be administered to a subject forinducing immune response against cancer in the subject by themselves orin combination with other drugs including the peptides, exosomes or CTLsof this invention. For example, the ex vivo administration can includethe steps of:

a: collecting APCs from a first subject;

b: contacting the APCs of step a with the peptide; and

c: administering the peptide-loaded APCs of step b to a second subject.

The first subject and the second subject can be the same individual, ormay be different individuals. The APCs obtained by step b can beadministered as a vaccine for treating and/or preventing cancerincluding hepatocellular carcinoma. In addition, the present inventionprovides a method or process for manufacturing a pharmaceuticalcomposition inducing antigen-presenting cells, wherein the methodcontains the step of admixing or formulating the peptide of the presentinvention with a pharmaceutically acceptable carrier.

According to an aspect of the present invention, the APCs have a highlevel of CTL inducibility. In the term of “high level of CTLinducibility”, the high level is relative to the level of that achievedby APCs contacted with no peptide or peptides which can not induce CTLs.Such APCs having a high level of CTL inducibility can be prepared by amethod which includes the step of transferring genes containing thepolynucleotides of this invention to APCs in vitro as well as by themethod mentioned above. The introduced genes can be in the form of DNAsor RNAs. Examples of methods for introduction include, withoutparticular limitations, various methods conventionally performed in thisfield, such as lipofection, electroporation, and calcium phosphatemethod can be used. More specifically, it can be performed as describedin Cancer Res 1996, 56: 5672-7; J Immunol 1998, 161: 5607-13; J Exp Med1996, 184: 465-72; Published Japanese Translation of InternationalPublication No. 2000-509281. By transferring the gene into APCs, thegene undergoes transcription, translation, and such in the cell, andthen the obtained protein is processed and presented by MHC Class I orClass II through a presentation pathway.

VII. Cytotoxic T Lymphocytes (CTLs)

A CTL induced against any of the peptides of the present inventionstrengthens the immune response targeting cancer cells in vivo and thuscan be used as vaccines, in a fashion similar to the peptides per se.Thus, the present invention also provides isolated CTLs that arespecifically induced or activated by any of the present peptides.

Such CTLs can be obtained by (1) administering the peptides of thepresent invention to a subject, collecting CTLs from the subject; (2)contacting (stimulating) subject-derived APCs and CD8-positive cells, orperipheral blood mononuclear leukocytes in vitro with the peptides ofthe present invention and then isolating CTLs; (3) contactingCD8-positive cells or peripheral blood mononuclear leukocytes in vitrowith the APCs or exosomes presenting a complex of an HLA antigen and apeptide of present invention on its surface and then isolating CTLs; or(4) introducing into a T cell, a gene encoding a T cell receptor (TCR)subunit polypeptide which binds to a peptide of this invention. The APCsor exosomes can be prepared by the methods described above, and themethod of (4) is detailed below under the section of “VIII. T cellreceptor (TCR)”.

The CTLs of this invention, which have been induced by stimulation withAPCs that present a peptide of this invention, can be derived frompatients who are subject to treatment and/or prevention, and can beadministered by themselves or in combination with other drugs includingthe peptides of this invention or exosomes for the purpose of regulatingeffects. The obtained CTLs act specifically against target cellspresenting the peptides of this invention, for example, the samepeptides used for induction. The target cells can be cells thatendogenously express WDRPUH, for example, hepatocellular carcinoma, orcells that are transfected with the WDRPUH gene; and cells that presenta peptide of this invention on the cell surface due to stimulation bythe peptide can also serve as targets of activated CTL attack.

VIII. T Cell Receptor (TCR)

The present invention also provides a composition containing nucleicacids encoding polypeptides that are capable of forming a subunit of a Tcell receptor (TCR), and methods of using the same. The TCR subunitshave the ability to form TCRs that confer specificity to T cells againsttumor cells presenting the specific peptide of the present invention. Byusing known methods in the art, the nucleic acids of alpha- andbeta-chains as the TCR subunits of the CTL induced with one or morepeptides of this invention can be identified (WO2007/032255 and Morganet al., J Immunol, 171, 3288 (2003)). For example, the PCR method ispreferred to analyze the TCR. The PCR primers for the analysis can be,for example,

5′-R primers (5′-gtctaccaggcattcgcttcat-3′) as 5′ side primers (SEQ IDNO: 65) and

3-TRa-C primers (5′-tcagctggaccacagccgcagcgt-3′) specific to TCR alphachain C region (SEQ ID NO: 66),

3-TRb-C1 primers (5′-tcagaaatcctttctcttgac-3′) specific to TCR betachain C1 region (SEQ ID NO: 67) or

3-TRbeta-C2 primers (5′-ctagcctctggaatcctttctctt-3′) specific to TCRbeta chain C2 region (SEQ ID NO: 68) as 3′ side primers,

but not limited. The derivative TCR can bind target cells displaying theWDRPUH peptide with high avidity, and optionally mediate efficientkilling of target cells presenting the WDRPUH peptide in vivo and invitro.

The nucleic acids encoding the TCR subunits can be incorporated intosuitable vectors e.g. retroviral vectors. These vectors are well knownin the art. The nucleic acids or the vectors containing them usefullycan be transferred into a T cell, for example, a T cell from a patient.Advantageously, the invention provides an off-the-shelf compositionallowing rapid modification of a patient's own T cells (or those ofanother mammal) to rapidly and easily produce modified T cells havingexcellent cancer cell killing properties.

The specific TCR is a receptor capable of specifically recognizing acomplex of a peptide of the present invention and HLA molecule, giving aT cell specific activity against the target cell when the TCR on thesurface of the T cell. A specific recognition of the above complex maybe confirmed by any known methods, and preferred methods include, forexample, tetramer analysis using HLA molecule and peptide of theinvention, and ELISPOT assay. By performing the ELISPOT assay, it can beconfirmed that a T cell expressing the TCR on the cell surfacerecognizes a cell by the TCR, and that the signal is transmittedintracellularly. The confirmation that the above-mentioned complex cangive a T cell cytotoxic activity when the complex exists on the T cellsurface may also be carried out by a known method. A preferred methodincludes, for example, the determination of cytotoxic activity againstan HLA positive target cell, such as chromium release assay. Also, thepresent invention provides CTLs which are prepared by transduction withthe nucleic acids encoding the TCR subunits polypeptides that bind tothe WDRPUH peptide of, e.g., SEQ ID NOs: 1, 2, 3, 4, 16 and 17 in thecontext of HLA-A2, and also the peptides of SEQ ID NOs: 30, 31, 34, 36,37, 40, 41, 45, 49, 55, 57 and 61 in the context of HLA-A24. Thetransduced CTLs are capable of homing to cancer cells in vivo, and canbe expanded by well known culturing methods in vitro (e.g., Kawakami etal., J Immunol., 142, 3452-3461 (1989)). The CTLs of the invention canbe used to form an immunogenic composition useful in the treatment orprophylaxis of cancer in a patient in need of therapy or protection(WO2006/031221).

Prevention and prophylaxis include any activity which reduces the burdenof mortality or morbidity from disease. Prevention and prophylaxis canoccur “at primary, secondary and tertiary prevention levels.” Whileprimary prevention and prophylaxis avoid the development of a disease,secondary and tertiary levels of prevention and prophylaxis encompassactivities aimed at the prevention and prophylaxis of the progression ofa disease and the emergence of symptoms as well as reducing the negativeimpact of an already established disease by restoring function andreducing disease-related complications. Alternatively, prevention andprophylaxis include a wide range of prophylactic therapies aimed atalleviating the severity of the particular disorder, e.g. reducing theproliferation and metastasis of tumors, reducing angiogenesis.

Treatment and/or prophylaxis of cancer or, and/or prevention ofpostoperative recurrence thereof includes any of the following steps,such as surgical removal of cancer cells, inhibition of the growth ofcancerous cells, involution or regression of a tumor, induction ofremission and suppression of occurrence of cancer, tumor regression, andreduction or inhibition of metastasis. Effective treatment and/or theprophylaxis of cancer decreases mortality and improves the prognosis ofindividuals having cancer, decreases the levels of tumor markers in theblood, and alleviates detectable symptoms accompanying cancer. Forexample, reduction or improvement of symptoms constitutes effectivetreatment and/or prophylaxis, and such reduction or improvement ofsymptoms include 10%, 20%, 30% or more reduction, or sustaining a stabledisease state.

IX. Pharmaceutical Agents or Compositions

Since WDRPUH expression is specifically elevated (up-regulated) inhepatocellular carcinoma as compared with normal tissue (Silva et al.,Neoplasia 2005 April; 7(4):348-55), the peptides or polynucleotides ofthe present invention can be used for the treatment and/or theprophylaxis of cancer or tumor, and/or prevention of postoperativerecurrence thereof. Thus, the present invention provides apharmaceutical agent or composition for the treatment and/or theprophylaxis of cancer or tumor, and/or prevention of postoperativerecurrence thereof, which includes one or more of the peptides, orpolynucleotides of this invention as an active ingredient.Alternatively, the present peptides can be expressed on the surface ofany of the foregoing exosomes or cells, such as APCs for the use aspharmaceutical agents or compositions. In addition, the aforementionedCTLs which target any of the peptides of the invention can also be usedas the active ingredient of the present pharmaceutical agents orcompositions.

In another embodiment, the present invention also provide the use of anactive ingredient selected from among:

(a) a peptide of the present invention;

(b) a nucleic acid encoding such a peptide as disclosed herein in anexpressible form;

(c) an APC or an exosome presenting a peptide of the present inventionon its surface; and

(d) a CTL of the present invention

in manufacturing a pharmaceutical composition or agent for treating orpreventing cancer or tumor.

Alternatively, the present invention further provides an activeingredient selected from among:

(a) a peptide of the present invention;

(b) a nucleic acid encoding such a peptide as disclosed herein in anexpressible form;

(c) an APC or an exosome presenting a peptide of the present inventionon its surface; and

(d) a CTL of the present invention

for use in treating or preventing cancer or tumor.

Alternatively, the present invention further provides a method orprocess for manufacturing a pharmaceutical composition or agent fortreating or preventing cancer or tumor, wherein the method or processincludes the step of formulating a pharmaceutically or physiologicallyacceptable carrier with an active ingredient selected from among:

(a) a peptide of the present invention;

(b) a nucleic acid encoding such a peptide as disclosed herein in anexpressible form;

(c) an APC or an exosome presenting a peptide of the present inventionon its surface; and

(d) a CTL of the present invention

as active ingredients.

In another embodiment, the present invention also provides a method orprocess for manufacturing a pharmaceutical composition or agent fortreating or preventing cancer or tumor, wherein the method or processincludes the step of admixing an active ingredient with apharmaceutically or physiologically acceptable carrier, wherein theactive ingredient is selected from among:

(a) a peptide of the present invention;

(b) a nucleic acid encoding such a peptide as disclosed herein in anexpressible form;

(c) an APC or an exosome presenting a peptide of the present inventionon its surface; and

(d) a CTL of the present invention.

Alternatively, the pharmaceutical composition or agent of the presentinvention may be used for either or both the prophylaxis of cancer ortumor and prevention of postoperative recurrence thereof.

The present pharmaceutical agents or compositions find use as a vaccine.In the context of the present invention, the phrase “vaccine” (alsoreferred to as an immunogenic composition) refers to a substance thathas the function to induce anti-tumor immunity upon inoculation intoanimals.

The pharmaceutical agents or compositions of the present invention canbe used to treat and/or prevent cancers or tumors, and/or prevention ofpostoperative recurrence thereof in subjects or patients including humanand any other mammal including, but not limited to, mouse, rat,guinea-pig, rabbit, cat, dog, sheep, goat, pig, cattle, horse, monkey,baboon, and chimpanzee, particularly a commercially important animal ora domesticated animal.

According to the present invention, peptides having an amino acidsequence selected from among SEQ ID NOs: 1, 2, 3, 4, 16, 17, 30, 31, 34,36, 37, 40, 41, 45, 49, 55, 57 and 61 have been found to be HLA-A24 orHLA-A2 restricted epitope peptides or candidates, respectively, that caninduce potent and specific immune response. Therefore, the presentpharmaceutical agents which include any of these peptides having theamino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 16, 17, 30, 31, 34, 36,37, 40, 41, 45, 49, 55, 57 and 61 are particularly suited for theadministration to subjects whose HLA antigen is HLA-A24 or HLA-A2.Especially, agents containing peptides having an amino acid sequence ofSEQ ID NOs: 1, 2, 3, 4, 16 and 17 are suited for the administration tosubjects of the HLA-A24 type, and those having an amino acid sequence ofSEQ ID NOs: 30, 31, 34, 36, 37, 40, 41, 45, 49, 55, 57 and 61 are suitedfor the administration to subjects of the HLA-A2 type. The same appliesto pharmaceutical agents and compositions which include polynucleotidesencoding any of these peptides (i.e. the polynucleotides of thisinvention).

Cancers or tumors to be treated by the pharmaceutical agents orcompositions of the present invention are not limited and include allkinds of cancers or tumors wherein WDRPUH is involved, for example,hepatocellular carcinoma.

The present pharmaceutical agents or compositions can contain inaddition to the aforementioned active ingredients, other peptides whichhave the ability to induce CTLs against cancerous cells, otherpolynucleotides encoding the other peptides, other cells that presentthe other peptides, or such. Herein, the other peptides that have theability to induce CTLs against cancerous cells are exemplified by cancerspecific antigens (e.g., identified TAAs), but are not limited thereto.

If needed, the pharmaceutical agents or compositions of the presentinvention can optionally include other therapeutic substances as anactive ingredient, so long as the substance does not inhibit theantitumoral effect of the active ingredient, e.g., any of the presentpeptides. For example, formulations can include anti-inflammatoryagents, pain killers, chemotherapeutics, and the like. In addition toincluding other therapeutic substances in the medicament itself, themedicaments of the present invention can also be administeredsequentially or concurrently with the one or more other pharmacologicagents. The amounts of medicament and pharmacologic agent depend, forexample, on what type of pharmacologic agent(s) is/are used, the diseasebeing treated, and the scheduling and routes of administration.

It should be understood that in addition to the ingredients particularlymentioned herein, the pharmaceutical agents or compositions of thisinvention can include other agents conventional in the art having regardto the type of formulation in question.

In one embodiment of the present invention, the present pharmaceuticalagents or compositions can be included in articles of manufacture andkits containing materials useful for treating the pathologicalconditions of the disease to be treated, e.g., cancer. The article ofmanufacture can include a container of any of the present pharmaceuticalagents with a label. Suitable containers include bottles, vials, andtest tubes. The containers can be formed from a variety of materials,such as glass or plastic. The label on the container should indicate theagent or composition is used for treating or preventing one or moreconditions of the disease. The label can also indicate directions foradministration and so on.

In addition to the container described above, a kit including apharmaceutical agent or composition of the present invention canoptionally further include a second container housing apharmaceutically-acceptable diluent. It can further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

The pharmaceutical agents or compositions can, if desired, be presentedin a pack or dispenser device which can contain one or more unit dosageforms containing the active ingredient. The pack can, for example,include metal or plastic foil, such as a blister pack. The pack ordispenser device can be accompanied by instructions for administration.

(1) Pharmaceutical Agents or Compositions Containing the Peptides as theActive Ingredient

The peptides of this invention can be administered directly as apharmaceutical agent or composition, or if necessary, that has beenformulated by conventional formulation methods. In the latter case, inaddition to the peptides of this invention, carriers, excipients, andsuch that are ordinarily used for drugs can be included as appropriatewithout particular limitations. Examples of such carriers are sterilizedwater, physiological saline, phosphate buffer, culture fluid and such.Furthermore, the pharmaceutical agents or compositions can contain asnecessary, stabilizers, suspensions, preservatives, surfactants andsuch. The pharmaceutical agents or compositions of this invention can beused for anticancer purposes.

The peptides of this invention can be prepared as a combination composedof two or more of the peptides of the present invention, to induce CTLsin vivo. The peptide combination can take the form of a cocktail or canbe conjugated to each other using standard techniques. For example, thepeptides can be chemically linked or expressed as a single fusionpolypeptide sequence. The peptides in the combination can be the same ordifferent. By administering the peptides of this invention, the peptidesare presented at a high density by the HLA antigens on APCs, then CTLsthat specifically react toward the complex formed between the displayedpeptide and the HLA antigen are induced. Alternatively, APCs thatpresent any of the peptides of this invention on their cell surface,which may be obtained by stimulating APCs (e.g., DCs) derived from asubject with the peptides of this invention may be administered to thesubject, and as a result, CTLs are induced in the subject andaggressiveness towards the cancer cells can be increased.

The pharmaceutical agents or composition for the treatment and/orprevention of cancer or tumor, which include a peptide of this inventionas the active ingredient, can also include an adjuvant known toeffectively establish cellular immunity. Alternatively, thepharmaceutical agents or compositions can be administered with otheractive ingredients or administered by formulation into granules. Anadjuvant refers to a compound that enhances the immune response againstthe protein when administered together (or successively) with theprotein having immunological activity. Adjuvants contemplated hereininclude those described in the literature (Clin Microbiol Rev 1994, 7:277-89). Examples of suitable adjuvants include aluminum phosphate,aluminum hydroxide, alum, cholera toxin, salmonella toxin, and such, butare not limited thereto.

Furthermore, liposome formulations, granular formulations in which thepeptide is bound to few-micrometers diameter beads, and formulations inwhich a lipid is bound to the peptide may be conveniently used.

In some embodiments, the pharmaceutical agents or compositions of thepresent invention may further include a component which primes CTLs.Lipids have been identified as agents capable of priming CTLs in vivoagainst viral antigens. For example, palmitic acid residues can beattached to the epsilon- and alpha-amino groups of a lysine residue andthen linked to a peptide of the present invention. The lipidated peptidecan then be administered either directly in a micelle or particle,incorporated into a liposome, or emulsified in an adjuvant. As anotherexample of lipid priming CTLs responses, E. coli lipoproteins, such astripalmitoyl-S-glycerylcysteinyl-seryl-serine (P3CSS) can be used toprime CTL when covalently attached to an appropriate peptide (see, e.g.,Deres et al., Nature 1989, 342: 561-4).

The method of administration can be oral, intradermal, subcutaneous,intravenous injection, or such, and systemic administration or localadministration to the vicinity of the targeted sites. The administrationcan be performed by single administration or boosted by multipleadministrations. The dose of the peptides of this invention can beadjusted appropriately according to the disease to be treated, age ofthe patient, weight, method of administration, and such, and isordinarily 0.001 mg to 1000 mg, for example, 0.001 mg to 1000 mg, forexample, 0.1 mg to 10 mg, and can be administered once in a few days tofew months. One skilled in the art can appropriately select a suitabledose.

(2) Pharmaceutical Agents or Compositions Containing Polynucleotides asthe Active Ingredient

The pharmaceutical agents or compositions of the present invention canalso contain nucleic acids encoding the peptides disclosed herein in anexpressible form. Herein, the phrase “in an expressible form” means thatthe polynucleotide, when introduced into a cell, will be expressed invivo as a polypeptide that induces anti-tumor immunity. In anexemplified embodiment, the nucleic acid sequence of the polynucleotideof interest includes regulatory elements necessary for expression of thepolynucleotide. The polynucleotide(s) can be equipped so to achievestable insertion into the genome of the target cell (see, e.g., Thomas KR & Capecchi M R, Cell 1987, 51: 503-12 for a description of homologousrecombination cassette vectors). See, e.g., Wolff et al., Science 1990,247: 1465-8; U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118;5,736,524; 5,679,647; and WO 98/04720. Examples of DNA-based deliverytechnologies include “naked DNA”, facilitated (bupivacaine, polymers,peptide-mediated) delivery, cationic lipid complexes, andparticle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g.,U.S. Pat. No. 5,922,687).

The peptides of the invention can also be expressed by viral orbacterial vectors. Examples of expression vectors include attenuatedviral hosts, such as vaccinia or fowlpox. This approach involves the useof vaccinia virus, e.g., as a vector to express nucleotide sequencesthat encode the peptide. Upon introduction into a host, the recombinantvaccinia virus expresses the immunogenic peptide, and thereby elicits animmune response. Vaccinia vectors and methods useful in immunizationprotocols are described in, e.g., U.S. Pat. No. 4,722,848. Anothervector is BCG (Bacille Calmette Guerin). BCG vectors are described inStover et al., Nature 1991, 351: 456-60. A wide variety of other vectorsuseful for therapeutic administration or immunization e.g., adeno andadeno-associated virus vectors, retroviral vectors, Salmonella typhivectors, detoxified anthrax toxin vectors, and the like, will beapparent. See, e.g., Shata et al., Mol Med Today 2000, 6: 66-71;Shedlock et al., J Leukoc Biol 2000, 68: 793-806; Hipp et al., In Vivo2000, 14: 571-85.

Delivery of a polynucleotide into a subject can be either direct, inwhich case the subject is directly exposed to a polynucleotide-carryingvector, or indirect, in which case, cells are first transformed with thepolynucleotide of interest in vitro, then the cells are transplantedinto the subject. These two approaches are known, respectively, as invivo and ex vivo gene therapies.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy 1993, 12: 488-505; Wu and Wu, Biotherapy 1991, 3:87-95; Tolstoshev, Ann Rev Pharmacol Toxicol 1993, 33: 573-96; Mulligan,Science 1993, 260: 926-32; Morgan & Anderson, Ann Rev Biochem 1993, 62:191-217; Trends in Biotechnology 1993, 11(5): 155-215). Methods commonlyknown in the art of recombinant DNA technology which can also be usedfor the present invention are described in eds. Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, NY, 1993; andKrieger, Gene Transfer and Expression, A Laboratory Manual, StocktonPress, NY, 1990.

The method of administration can be oral, intradermal, subcutaneous,intravenous injection, or such, and systemic administration or localadministration to the vicinity of the targeted sites finds use. Theadministration can be performed by single administration or boosted bymultiple administrations. The dose of the polynucleotide in the suitablecarrier or cells transformed with the polynucleotide encoding thepeptides of this invention can be adjusted appropriately according tothe disease to be treated, age of the patient, weight, method ofadministration, and such, and is ordinarily 0.001 mg to 1000 mg, forexample, 0.001 mg to 1000 mg, for example, 0.1 mg to 10 mg, and can beadministered once every few days to once every few months. One skilledin the art can appropriately select the suitable dose.

X. Methods Using the Peptides, Exosomes, APCs and CTLs

The peptides and polynucleotides of the present invention can be usedfor inducing APCs and CTLs. The exosomes and APCs of the presentinvention can be also used for inducing CTLs. The peptides,polynucleotides, exosomes and APCs can be used in combination with anyother compounds so long as the compounds do not inhibit their CTLinducibility. Thus, any of the aforementioned pharmaceutical agents orcompositions of the present invention can be used for inducing CTLs, andin addition thereto, those including the peptides and polynucleotidescan be also be used for inducing APCs as discussed below.

(1) Method of Inducing Antigen-Presenting Cells (APCs)

The present invention provides methods of inducing APCs with high CTLinducibility using the peptides or polynucleotides of this invention.

The methods of the present invention contain the step of contacting APCswith the peptides of this invention in vitro, ex vivo or in vivo. Forexample, the method contacting APCs with the peptides ex vivo caninclude the steps of:

a: collecting APCs from a subject; and

b: contacting the APCs of step a with the peptide.

The APCs are not limited to a particular kind of cells and include DCs,Langerhans cells, macrophages, B cells, and activated T cells, which areknown to present proteinaceous antigens on their cell surface so as tobe recognized by lymphocytes. Preferably, DCs can be used since theyhave the strongest CTL inducibility among APCs. Any peptides of thepresent invention can be used by themselves or with other peptides ofthis invention.

Alternatively, APCs may be induced by administering a peptide of thepresent invention to a subject to contact the peptide with APCs in vivo,and consequently, induce APCs with high CTL inducibility in the body ofthe subject. Thus, the present invention also contemplates administeringthe peptides of this invention to a subject for inducing APCs. Asanother method, a polynucleotide of this invention may be administeredto a subject in an expressible form to express a peptide of thisinvention and contact the peptide with APCs in vivo. Similarly to theadministration of a peptide of the present invention, APCs with high CTLinducibility are induced in the body of the subject. Thus, the presentinvention also contemplates administering the polynucleotides of thisinvention to a subject for inducing APCs. For the explanation on thephrase of “expressible form”, see section “IX. Pharmaceutical agents (2)Pharmaceutical agents containing polynucleotides as the activeingredient”.

Furthermore, the present invention also contemplates introducing apolypeptide of this invention into an APC to induce APC with CTLinducibility. For example, the method may include the steps of:

a: collecting an APC from a subject; and

b: introducing a polynucleotide encoding a peptide of this inventioninto the collected APC.

The step b can be performed as described above in section “VI.Antigen-presenting cells”.

(2) Method of Inducing CTLs

Furthermore, the present invention provides methods for inducing CTLsusing the peptides, polynucleotides, or exosomes or APCs of thisinvention.

Upon the administration of the peptides, the polynucleotides, APCs, orexosomes of this invention to a subject, CTLs are induced in the body ofthe subject to strengthen the immune response targeting cancer cells.Thus, it is another object of the present invention to provide a methodfor inducing CTLs, which method may include the step of administeringthe peptides, the polynucleotides, the APCs or exosomes of thisinvention to a subject.Alternatively, CTLs can be also induced ex vivo, and after theinduction, the activated CTLs may be returned to the subject. Forexample, the method may include the steps of:a: collecting APCs from a subject;b: contacting the APCs of step a with a peptide of the presentinvention; andc: co-culturing the APCs of step b with CD8-positive cells.

The APCs to be co-cultured with the CD8-positive cells in above step ccan also be prepared by transferring a polynucleotide encoding thepeptide of this invention into APCs as described above in section “VI.Antigen-presenting cells”; but are not limited thereto and any APCswhich effectively presents, on its surface, a complex of an HLA antigenand a peptide of this invention may be used for the present method.

In place of such APCs, exosomes that present on their surface a complexof an HLA antigen and a peptide of this invention may be also used.Namely, the present inventive method for inducing CTLs may include thestep of co-culturing exosomes presenting on its surface a complex of anHLA antigen and a peptide of this invention. Such exosomes may beprepared by the methods described above in section “V. Exosomes”.Furthermore, CTLs can be induced by introducing a polynucleotideencoding a TCR subunit binding to a peptide of this invention intoCD8-positive cells. Such transduction may be performed as describedabove in section “VIII. T cell receptor (TCR)”.

(3) Method of Inducing Immune Response

The present invention further provides methods for inducing an immuneresponse against cancer in a subject. The methods include theadministration of a vaccine of the present invention, which contains:

(a) one or more peptides of the present invention, or an immunologicallyactive fragment thereof;

(b) one or more polynucleotides encoding the peptides or theimmunologically active fragment of (a);

(c) one or more isolated CTLs of the present invention; or

(d) one or more isolated antigen-presenting cells of the presentinvention.

In the present invention, cancer overexpressing WDRPUH can be treatedwith these active ingredients. Accordingly, prior to the administrationof the vaccines or pharmaceutical compositions comprising the activeingredients, it is preferable to confirm whether the expression level ofWDRPUH in the cancer cells or tissues to be treated is enhanced comparedwith normal cells of the same organ. Thus, in one embodiment, thepresent invention provides a method for treating cancer expressingWDRPUH, which method may include the steps of:

i) determining the expression level of WDRPUH in cancer cells or tissueobtained from a subject with the cancer to be treated;

ii) comparing the expression level of WDRPUH with normal control; and

iii) administrating at least one component selected from the groupconsisting of (a) to (d) described above to a subject with canceroverexpressing WDRPUH compared with normal control.

Alternatively, the present invention also provides a vaccine orpharmaceutical composition comprising at least one component selectedfrom the group consisting of (a) to (d) described above, for use inadministrating to a subject having cancer overexpressing WDRPUH. Inother words, the present invention further provides a method foridentifying a subject to be treated with the WDRPUH polypeptide of thepresent invention, which method may include the step of determining anexpression level of WDRPUH in subject-derived cancer cells or tissue,wherein an increase of the level compared to a normal control level ofthe gene indicates that the subject has cancer which may be treated withthe WDRPUH polypeptide of the present invention. The method of treatingcancer of the present invention will be described in more detail below.

A subject to be treated by the present method is preferably a mammal.Exemplary mammals include, but are not limited to, e.g., human,non-human primate, mouse, rat, dog, cat, horse, and cow.

According to the present invention, the expression level of WDRPUH inthe cancer cells or tissues obtained from a subject is determined. Theexpression level can be determined at the transcription (nucleic acid)product level, using methods known in the art. For example, the mRNA ofWDRPUH may be quantified using probes by hybridization methods (e.g.,Northern hybridization). The detection may be carried out on a chip oran array. The use of an array is preferable for detecting the expressionlevel of WDRPUH. Those skilled in the art can prepare such probesutilizing the sequence information of WDRPUH. For example, the cDNA ofWDRPUH may be used as the probes. If necessary, the probe may be labeledwith a suitable label, such as dyes, fluorescent substances andisotopes, and the expression level of the gene may be detected as theintensity of the hybridized labels.

Furthermore, the transcription product of WDRPUH (SEQ ID NO: 63) may bequantified using primers by amplification-based detection methods (e.g.,RT-PCR). Such primers can also be prepared based on the availablesequence information of the gene.

Specifically, a probe or primer used for the present method hybridizesunder stringent, moderately stringent, or low stringent conditions tothe mRNA of WDRPUH. As used herein, the phrase “stringent(hybridization) conditions” refers to conditions under which a probe orprimer will hybridize to its target sequence, but not to othersequences. Stringent conditions are sequence-dependent and will bedifferent under different circumstances. Specific hybridization oflonger sequences is observed at higher temperatures than shortersequences. Generally, the temperature of a stringent condition isselected to be about 5 degree Centigrade lower than the thermal meltingpoint (Tm) for a specific sequence at a defined ionic strength and pH.The Tm is the temperature (under a defined ionic strength, pH andnucleic acid concentration) at which 50% of the probes complementary tothe target sequence hybridize to the target sequence at equilibrium.Since the target sequences are generally present at excess, at Tm, 50%of the probes are occupied at equilibrium. Typically, stringentconditions will be those in which the salt concentration is less thanabout 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30degree Centigrade for short probes or primers (e.g., 10 to 50nucleotides) and at least about 60 degree Centigrade for longer probesor primers. Stringent conditions may also be achieved with the additionof destabilizing agents, such as formamide.

Alternatively, the translation product may be detected for the diagnosisof the present invention. For example, the quantity of WDRPUH protein(SEQ ID NO: 64) may be determined. Methods for determining the quantityof the protein as the translation product include immunoassay methodsthat use an antibody specifically recognizing the protein. The antibodymay be monoclonal or polyclonal. Furthermore, any fragment ormodification (e.g., chimeric antibody, scFv, Fab, F(ab′)2, Fv, etc.) ofthe antibody may be used for the detection, so long as the fragment ormodified antibody retains the binding ability to WDRPUH protein. Methodsto prepare these kinds of antibodies for the detection of proteins arewell known in the art, and any method may be employed in the presentinvention to prepare such antibodies and equivalents thereof.

As another method to detect the expression level of WDRPUH gene based onits translation product, the intensity of staining may be observed viaimmunohistochemical analysis using an antibody against WDRPUH protein.Namely, the observation of strong staining indicates increased presenceof the protein and, at the same time, high expression level of WDRPUHgene.

The expression level of target gene including WDRPUH gene in cancercells can be considered to be increased if it increases from the controllevel of the corresponding the target gene by, for example, 10%, 25%, or50%; or increases to more than 1.1 fold, more than 1.5 fold, more than2.0 fold, more than 5.0 fold, more than 10.0 fold, or more.

The control level may be determined at the same time with the cancercells by using a sample(s) previously collected and stored from asubject/subjects whose disease state(s) (cancerous or non-cancerous)is/are known. In addition, normal cells obtained from non-cancerousregions of an organ that has the cancer to be treated may be used asnormal control. Alternatively, the control level may be determined by astatistical method based on the results obtained by analyzing previouslydetermined expression level(s) of WDRPUH gene in samples from subjectswhose disease states are known. Furthermore, the control level can be adatabase of expression patterns from previously tested cells.

Moreover, according to an aspect of the present invention, theexpression level of WDRPUH gene in a biological sample may be comparedto multiple control levels, which control levels are determined frommultiple reference samples. It is preferred to use a control leveldetermined from a reference sample derived from a tissue type similar tothat of the subject-derived biological sample. Moreover, it ispreferred, to use the standard value of the expression levels of WDRPUHgene in a population with a known disease state. The standard value maybe obtained by any method known in the art. For example, a range ofmean+/−2 S.D. or mean+/−3 S.D. may be used as the standard value.

In the context of the present invention, a control level determined froma biological sample that is known to be non-cancerous is referred to asa “normal control level”. On the other hand, if the control level isdetermined from a cancerous biological sample, it is referred to as a“cancerous control level”.

When the expression level of WDRPUH gene is increased as compared to thenormal control level or is similar to the cancerous control level, thesubject may be diagnosed with cancer to be treated.

The present invention also provides a kit for determining a subjectsuffering from cancer which can be treated with the WDRPUH polypeptideof the present invention, which may also be useful in assessing and/ormonitoring the efficacy of a cancer immunotherapy. Preferably, thecancer is hepatocellular carcinoma. More particularly, the kitpreferably includes at least one reagent for detecting the expression ofthe WDRPUH gene in a subject-derived cancer cell, which reagent may beselected from the group of:

(a) a reagent for detecting mRNA of the WDRPUH gene;

(b) a reagent for detecting the WDRPUH protein; and

(c) a reagent for detecting the biological activity of the WDRPUHprotein.

Suitable reagents for detecting mRNA of the WDRPUH gene include nucleicacids that specifically bind to or identify the WDRPUH mRNA, such asoligonucleotides which have a complementary sequence to a part of theWDRPUH mRNA. These kinds of oligonucleotides are exemplified by primersand probes that are specific to the WDRPUH mRNA. These kinds ofoligonucleotides may be prepared based on methods well known in the art.If needed, the reagent for detecting the WDRPUH mRNA may be immobilizedon a solid matrix. Moreover, more than one reagent for detecting theWDRPUH mRNA may be included in the kit.

On the other hand, suitable reagents for detecting the WDRPUH proteininclude antibodies to the WDRPUH protein. The antibody may be monoclonalor polyclonal. Furthermore, any fragment or modification (e.g., chimericantibody, scFv, Fab, F(ab′)2, Fv, etc.) of the antibody may be used asthe reagent, so long as the fragment or modified antibody retains thebinding ability to the WDRPUH protein. Methods to prepare these kinds ofantibodies for the detection of proteins are well known in the art, andany method may be employed in the present invention to prepare suchantibodies and equivalents thereof. Furthermore, the antibody may belabeled with signal generating molecules via direct linkage or anindirect labeling technique. Labels and methods for labeling antibodiesand detecting the binding of antibodies to their targets are well knownin the art, and any labels and methods may be employed for the presentinvention. Moreover, more than one reagent for detecting the WDRPUHprotein may be included in the kit.

The kit may contain more than one of the aforementioned reagents. Forexample, tissue samples obtained from subjects suffering from cancer ornot may serve as useful control reagents. A kit of the present inventionmay further include other materials desirable from a commercial and userstandpoint, including buffers, diluents, filters, needles, syringes, andpackage inserts (e.g., written, tape, CD-ROM, etc.) with instructionsfor use. These reagents and such may be retained in a container with alabel. Suitable containers include bottles, vials, and test tubes. Thecontainers may be formed from a variety of materials, such as glass orplastic.

As an embodiment of the present invention, when the reagent is a probeagainst the WDRPUH mRNA, the reagent may be immobilized on a solidmatrix, such as a porous strip, to form at least one detection site. Themeasurement or detection region of the porous strip may include aplurality of sites, each containing a nucleic acid (probe). A test stripmay also contain sites for negative and/or positive controls.Alternatively, control sites may be located on a strip separated fromthe test strip. Optionally, the different detection sites may containdifferent amounts of immobilized nucleic acids, i.e., a higher amount inthe first detection site and lesser amounts in subsequent sites. Uponthe addition of test sample, the number of sites displaying a detectablesignal provides a quantitative indication of the amount of WDRPUH mRNApresent in the sample. The detection sites may be configured in anysuitably detectable shape and are typically in the shape of a bar or dotspanning the width of a test strip.

The kit of the present invention may further include a positive controlsample or WDRPUH standard sample. The positive control sample of thepresent invention may be prepared by collecting WDRPUH positive samplesand then those WDRPUH level are assayed. Alternatively, purified WDRPUHprotein or polynucleotide may be added to cells non-expressing WDRPUH toform the positive sample or the WDRPUH standard. In the presentinvention, purified WDRPUH may be recombinant protein. The WDRPUH levelof the positive control sample is, for example, more than the cut offvalue.

The following examples are presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of theinvention.

EXAMPLES

Materials and Methods

Cell Lines

A24 lymphoblastoid cell line (A24LCL) was established by transformationwith Epstein-bar virus into HLA-A24 positive human B lymphocyte. T2(HLA-A2), COS 7, African green monkey kidney cell line, were purchasedfrom ATCC.

Candidate Selection of Peptides Derived from WDRPUH

9-mer and 10-mer peptides derived from WDRPUH that bind to HLA-A*2402and HLA-A*0201 molecules were predicted using binding predictionsoftware “BIMAS” (http://www-bimas.cit.nih.gov/molbio/hla_bind) (Parkeret al. (J Immunol 1994, 152(1): 163-75), Kuzushima et al. (Blood 2001,98(6): 1872-81)). These peptides were synthesized by Sigma (Sapporo,Japan) or Biosynthesis Inc. (Lewisville, Tex.) according to a standardsolid phase synthesis method and purified by reversed phase highperformance liquid chromatography (HPLC). The purity (>90%) and theidentity of the peptides were determined by analytical HPLC and massspectrometry analysis, respectively. Peptides were dissolved indimethylsulfoxide (DMSO) at 20 mg/ml and stored at −80 degrees C.

In Vitro CTL Induction

Monocyte-derived dendritic cells (DCs) were used as antigen-presentingcells (APCs) to induce cytotoxic T lymphocyte (CTL) responses againstpeptides presented on human leukocyte antigen (HLA). DCs were generatedin vitro as described elsewhere (Nakahara S et al., Cancer Res 2003 Jul.15, 63(14): 4112-8). Specifically, peripheral blood mononuclear cells(PBMCs) isolated from a normal volunteer (HLA-A*2402 positive andHLA-A*0201 positive) by Ficoll-Plaque (Pharmacia) solution wereseparated by adherence to a plastic tissue culture dish (BectonDickinson) so as to enrich them as the monocyte fraction. Themonocyte-enriched population was cultured in the presence of 1000 U/mlof granulocyte-macrophage colony-stimulating factor (GM-CSF) (R&DSystem) and 1000 U/ml of interleukin (IL)-4 (R&D System) in AIM-V Medium(Invitrogen) containing 2% heat-inactivated autologous serum (AS). After7 days of culture, the cytokine-induced DCs were pulsed with 20micro-g/ml of each of the synthesized peptides in the presence of 3micro-g/ml of beta 2-microglobulin for 3 hrs at 37 degrees C. in AIM-VMedium. The generated cells appeared to express DC-associated molecules,such as CD80, CD83, CD86 and HLA class II, on their cell surfaces (datanot shown).

These peptide-pulsed DCs were then inactivated by Mitomycin C (MMC) (30micro-g/ml for 30 min) or X-irradiation (20 Gy) and mixed at a 1:20ratio with autologous CD8+ T cells, obtained by positive selection withCD8 Positive Isolation Kit (Dynal). These cultures were set up in48-well plates (Corning); each well contained 1.5×10⁴ peptide-pulsedDCs, 3×10⁵ CD8+ T cells and 10 ng/ml of IL-7 (R&D System) in 0.5 ml ofAIM-V/2% AS medium. Three days later, these cultures were supplementedwith IL-2 (CHIRON) to a final concentration of 20 IU/ml. On days 7 and14, the T cells were further stimulated with the autologouspeptide-pulsed DCs. The DCs were prepared each time following the samesteps as described above. CTLs were tested against peptide-pulsed A24LCLor T2 cells after the 3rd round of peptide stimulation on day 21 (TanakaH et al., Br J Cancer 2001 Jan. 5, 84(1): 94-9; Umano Y et al., Br JCancer 2001 Apr. 20, 84(8): 1052-7; Uchida N et al., Clin Cancer Res2004 Dec. 15, 10(24): 8577-86; Suda T et al., Cancer Sci 2006 May,97(5): 411-9; Watanabe T et al., Cancer Sci 2005 August, 96(8):498-506).

CTL Expansion Procedure

CTLs were expanded in culture using the method similar to the onedescribed by Riddell et al. (Walter E A et al., N Engl J Med 1995 Oct.19, 333(16): 1038-44; Riddell S R et al., Nat Med 1996 February, 2(2):216-23). A total of 5×10⁴ CTLs were suspended in 25 ml of AIM-V/5% ASmedium with 2 kinds of human B-lymphoblastoid cell lines, inactivated byMitomycin C, in the presence of 40 ng/ml of anti-CD3 monoclonal antibody(Pharmingen). One day after initiating the cultures, 120 IU/ml of IL-2were added to the cultures. The cultures were fed with fresh AIM-V/5% ASmedium containing 30 IU/ml of IL-2 on days 5, 8 and 11 (Tanaka H et al.,Br J Cancer 2001 Jan. 5, 84(1): 94-9; Umano Y et al., Br J Cancer 2001Apr. 20, 84(8): 1052-7; Uchida N et al., Clin Cancer Res 2004 Dec. 15,10(24): 8577-86; Suda T et al., Cancer Sci 2006 May, 97(5): 411-9;Watanabe T et al., Cancer Sci 2005 August, 96(8): 498-506).

Establishment of CTL Clones

The dilutions were made to have 0.3, 1, and 3 CTLs/well in 96round-bottomed micro titer plate (Nalge Nunc International). CTLs werecultured with 1×10⁴ cells/well of 2 kinds of human B-lymphoblastoid celllines, 30 ng/ml of anti-CD3 antibody, and 125 U/ml of IL-2 in a total of150 micro-l/well of AIM-V Medium containing 5% AS. 50 micro-1/well ofIL-2 were added to the medium 10 days later so as to reach a finalconcentration of 125 U/ml IL-2. CTL activity was tested on the 14th day,and CTL clones were expanded using the same method as described above(Uchida N et al., Clin Cancer Res 2004 Dec. 15, 10(24): 8577-86; Suda Tet al., Cancer Sci 2006 May, 97(5): 411-9; Watanabe T et al., Cancer Sci2005 August, 96(8): 498-506).

Specific CTL Activity

To examine specific CTL activity, interferon (IFN)-gamma enzyme-linkedimmunospot (ELISPOT) assay and IFN-gamma enzyme-linked immunosorbentassay (ELISA) were performed. Specifically, peptide-pulsed A24LCL(1×10⁴/well) or T2 (1×10⁴/well) was prepared as stimulator cells.Cultured cells in 48 wells were used as responder cells. IFN-gammaELISPOT assay and IFN-gamma ELISA assay were performed according to themanufacture's recommended procedure.

Plasmid Transfection

The cDNAs encoding the open reading frames of target genes, HLA-A24 andHLA-A*0201 were amplified by PCR. The PCR-amplified product of Targetgenes and HLA-A24 or HLA-A*0201 were cloned into pIRES vector (ClontechLaboratories, Inc., Cat. No. 631605). The plasmids were transfected intoCOS 7, which is a target genes and HLA-A24-null cell line, usinglipofectamine 2000 (Invitrogen) according to the manufacturer'srecommended procedures. After 2 days from transfection, the transfectedcells were harvested with versene (Invitrogen) and used as the targetcells (5×10⁴ cells/well) for CTL activity assay.

Results

Prediction of HLA-A24 and HLA-A2 Binding Peptides Derived from WDRPUH

Table 1 shows the HLA-A24 binding peptides of WDRPUH in order of highestbinding affinity. A total of 25 peptides having potential HLA-A24binding ability were selected and examined to determine the epitopepeptides (Table 1) Table 2 shows the HLA-A2 binding 9mer and 10merpeptides of WDRPUH in order of highest binding affinity. A total of 37peptides having potential HLA-A2 binding ability were selected andexamined to determine the epitope peptides (Table 2).

TABLE 1 HLA-A24 binding peptides derived from WDRPUH predicted by BIMASPeptide Start Amino Acid Binding SEQ ID name Position sequence Score NO.WDRPUH- 40 IYPLGCTVL 300 1 A24-9mer 314 IYRVSFTDF 120 2 509 CYHPEEFQI 603 339 VFPFGTAEL 33 4 318 SFTDFKETL 24 5 400 AFAPETGRL 24 6 118 AFSPNDLYL24 7 231 KMNPRTKLL 14.4 8 257 RCLKMGGLL 12 9 99 KNRELLARL 11.52 10 527AYWEVFDGT 10.08 11 248 KFSLGVSAI 10 12 WDRPUH- 280 GYKPiKKIQL 240 13A24-10mer 77 EYIAsGQVTF 150 14 509 CYHPeEFQII 86.4 15 409 MYVInNAHRI 7516 40 IYPLgCTVLI 75 17 220 FYLGtTTGDI 75 18 21 GFNGhVPTGL 42 19 531VFDGtVIREL 30.8 20 559 HFVTgGNDHL 30 21 495 RNQMiLANTL 17.28 22 339VFPFgTAELF 15 23 165 IFSRcRDEMF 10 24 331 HFDAvEDIVF 10 25 Startposition indicates the number of amino acid residues from the N-terminusof WDRPUH. Binding score is derived from “BIMAS”.

TABLE 2 HLA-A2 binding peptides derived from WDRPUH predicted by BIMASPeptide Start Amino Acid Binding SEQ ID name Position sequence Score NO.WDRPUH- 59 FLQGHGNNV 319.939 26 A2-9mer 499 ILANTLFQC 112.664 27 553ITQEGVHFV 85.173 28 231 KMNPRTKLL 53.999 29 39 SHYPLGCTV 52.025 30 407KLMYVINNA 42.278 31 264 LLVGSGAGL 36.316 32 193 KIWPTECQT 29.766 33 288QLQGGITSI 23.995 34 116 ALAFSPNDL 21.362 35 237 KLLTDVGPA 19.236 36 543SLSGSINGM 11.426 37 WDRPUH- 94 ILWDyKNREL 247.167 38 A2-10mer 490ILANtLFQCV 224.653 39 570 KVWDyNEGEV 94.23 40 263 GLLVgSGAGL 79.041 41155 GLNVgNATNV 69.552 42 498 MILAnTLFQC 57.318 43 305 FLVGtEESHI 47.99144 78 YIASgQVTFM 39.75 45 610 AILRwKYPYT 31.277 46 86 FMGFKADIIL 29.09847 325 TLIAtCHFDA 28.814 48 10 QVAEIELDAV 28.121 49 560 FVTGgNDHLV27.995 50 108 SLHKgKIEAL 24.075 51 374 NMTChGIDFM 22.24 52 221YLGTiTGDIL 19.742 53 231 KMNPrTKLLT 18.837 54 411 VINNaHRIGV 16.258 5551 AINTkEQNFL 16.155 56 287 IQLQgGITSI 15.303 57 326 LIATcHFDAV 15.13658 437 GEGEvRVWQI 14.347 59 338 IVFPfGTAEL 11.757 60 265 LVGSgAGLLV10.346 61 117 LAFSpNDLYL 10.264 62 Start position indicates the numberof amino acid residues from the N-terminus of WDRPUH. Binding score isderived from “BIMAS”.CTL Induction with the Predicted Peptides from WDRPUH Restricted withHLA-A*2402 and Establishment for CTL Lines Stimulated with WDRPUHDerived Peptides

CTLs for those peptides derived from WDRPUH were generated according tothe protocols as described in “Materials and Methods”. Peptide specificCTL activity was determined by IFN-gamma ELISPOT assay (FIG. 1a-f ). Itshowed that #3 and #6 stimulated with WDRPUH-A24-9-40 (SEQ ID NO: 1)(a), #8 with WDRPUH-A24-9-314 (SEQ ID NO: 2) (b), #2 and #6 withWDRPUH-A24-9-509 (SEQ ID NO: 3) (c), #1, #2 and #5 with WDRPUH-A24-9-339(SEQ ID NO: 4) (d), #2, #3, #4, #6, #7 and #8 with WDRPUH-A24-10-409(SEQ ID NO: 16) (e) and #5, #6 and #8 with WDRPUH-A24-10-40 (SEQ ID NO:17) (f) demonstrated potent IFN-gamma production as compared to thecontrol wells.

Furthermore, the cells in the positive well numbers #6 stimulated withSEQ ID NO: 1, #8 with SEQ ID NO: 2, #2 with SEQ ID NO: 3, #5 with SEQ IDNO: 4, #4 with SEQ ID NO: 16 and #6 with SEQ ID NO: 17 were expanded andestablished as CTL lines. CTL activity of these CTL lines was determinedby IFN-gamma ELISA assay (FIG. 2a-f ). All CTL lines demonstrated potentIFN-gamma production against the target cells pulsed with correspondingpeptides as compared to target cells without peptide pulse. On the otherhand, no CTL lines could be established by stimulation with otherpeptides shown in Table 1, despite the fact that the peptides werepredicted to have a binding activity with HLA-A*2402 (data not shown).As a result, 6 peptides derived from WDRPUH were screened as peptidesthat can induce potent CTL lines.

Specific CTL Activity Against Target Cells Exogenously Expressing WDRPUHand HLA-A*2402

The established CTL lines raised against the peptides of the presentinvention were examined for their ability to recognize target cells thatendogenously express WDRPUH and HLA-A*2402 molecule. Specific CTLactivity against COS 7 cells transfected with both the full length ofWDRPUH and HLA-A*2402 molecule genes (a specific model for the targetcells that exogenously express WDRPUH and HLA-A*2402 gene) was testedusing the CTL lines raised by corresponding peptides as the effectercells. COS 7 cells transfected with either the full length of WDRPUH orHLA-A* 2402 gene were prepared as control. In FIG. 3, the CTLsstimulated with SEQ ID NO: 2 showed potent CTL activity against COS 7cells expressing both WDRPUH and HLA-A* 2402. In contrast, nosignificant specific CTL activity was detected against the controls.Thus, these data clearly demonstrate that WDRPUH-A24-9-314 (SEQ ID NO:2) was naturally processed and presented on the target cell's surfacewith HLA-A*2402 molecule and was recognized by CTLs. These resultsindicated that this peptide derived from WDRPUH may be applicable ascancer vaccines for patients with WDRPUH expressing tumors.

CTL Induction with the Predicted Peptides from WDRPUH Restricted withHLA-A*0201

CTLs recognizing peptides derived from WDRPUH were generated accordingto the protocols as described in “Materials and Methods”. Peptidespecific CTL activity was determined by IFN-gamma ELISPOT assay (FIG. 4a-1). The well numbers #2 and #7 stimulated with WDRPUH-A2-9-39 (SEQ IDNO: 30) (a), #2 with WDRPUH-A2-9-407 (SEQ ID NO: 31) (b), #3 withWDRPUH-A2-9-288 (SEQ ID NO: 34) (c), #6 with WDRPUH-A2-9-237 (SEQ ID NO:36) (d), #4 with WDRPUH-A2-9-543 (SEQ ID NO: 37) (e), #4 withWDRPUH-A2-10-570 (SEQ ID NO: 40) (f), #2 and #8 with WDRPUH-A2-10-263(SEQ ID NO: 41) (g), #5 with WDRPUH-A2-10-78 (SEQ ID NO: 45) (h), #2with WDRPUH-A2-10-10 (SEQ ID NO: 49) (i), #6 with WDRPUH-A2-10-411 (SEQID NO: 55) (j), #7 with WDRPUH-A2-10-287 (SEQ ID NO: 57) (k) and #6 withWDRPUH-A2-10-265 (SEQ ID NO: 61) (1) demonstrated potent IFN-gammaproduction as compared to the control wells. On the other hand, nopotent IFN-gamma production could be detected by stimulation with otherpeptides shown in Table 2, despite the fact that these peptides werepredicted to have a binding activity with HLA-A*0201 (data not shown).

Establishment of CTL Lines and Clones Against WDRPUH Specific Peptides

The cells that showed peptide specific CTL activity by IFN-gamma ELISPOTassay in the well numbers #7 stimulated with SEQ ID NO: 30 and #3 withSEQ ID NO: 34 were expanded and established as CTL lines. CTL activityof these CTL lines was determined by IFN-gamma ELISA assay (FIGS. 5a andb ). Both CTL lines demonstrated potent IFN-gamma production against thetarget cells pulsed with corresponding peptides as compared to thetarget cells without peptide pulse. Furthermore, CTL clones wereestablished by limiting dilution from the CTL lines, and IFN-gammaproduction from the CTL clones against target cells pulsed with thecorresponding peptides was determined by IFN-gamma ELISA assay. PotentIFN-gamma productions from CTL clones stimulated with SEQ ID NO: 30 andSEQ ID NO: 34 are demonstrated in FIGS. 5c and d.

Specific CTL Activity Against Target Cells Exogenously Expressing WDRPUHand HLA-A*0201

The established CTL clones raised against the peptides of presentinvention were examined for their ability to recognize target cells thatendogenously express WDRPUH and HLA-A*0201 molecule. Specific CTLactivity against COS 7 cells transfected with both the full length ofWDRPUH and HLA-A*0201 molecule genes (a specific model for the targetcells that endogenously express WDRPUH and HLA-A*0201 gene) was testedusing the CTL lines raised by corresponding peptides as the effectorcells. COS 7 cells transfected with either the full length of WDRPUH orHLA-A* 0201 genes were prepared as controls. In FIG. 5e , the CTLsstimulated with SEQ ID NO: 34 showed potent CTL activity against COS 7cells expressing both WDRPUH and HLA-A* 0201. In contrast, nosignificant specific CTL activity was detected against the controls.These data clearly demonstrate that the peptides of WDRPUH-A02-9-288(SEQ ID NO: 34) were endogenously processed and presented on the targetcell's surface with HLA-A*0201 molecule and were recognized by the CTLs.These results indicated that WDRPUH-A02-9-288 (SEQ ID NO: 34) may beapplicable as cancer vaccines for patients with WDRPUH expressingtumors.

Homology Analysis of Antigen Peptides

The CTLs stimulated with

WDRPUH-A24-9-40, (SEQ ID NO: 1) WDRPUH-A24-9-314, (SEQ ID NO: 2)WDRPUH-A24-9-509, (SEQ ID NO: 3) WDRPUH-A24-9-339, (SEQ ID NO: 4)WDRPUH-A24-10-409, (SEQ ID NO: 16) WDRPUH-A24-10-40, (SEQ ID NO: 17)WDRPUH-A02-9-39, (SEQ ID NO: 30) WDRPUH-A02-9-407, (SEQ ID NO: 31)WDRPUH-A02-9-288, (SEQ ID NO: 34) WDRPUH-A02-9-237, (SEQ ID NO: 36)WDRPUH-A02-9-543, (SEQ ID NO: 37) WDRPUH-A02-10-570, (SEQ ID NO: 40)WDRPUH-A02-10-263, (SEQ ID NO: 41) WDRPUH-A02-10-78, (SEQ ID NO: 45)WDRPUH-A02-10-10, (SEQ ID NO: 49) WDRPUH-A02-10-411, (SEQ ID NO: 55)WDRPUH-A02-10-287  (SEQ ID NO: 57) and WDRPUH-A02-10-265 (SEQ ID NO: 61)showed significant and specific CTL activity. This result may be due tothe fact that these peptide sequences are homologous to peptides derivedfrom other molecules that are known to sensitize the human immunesystem.

To exclude this possibility, homology analyses were performed for thesepeptide sequences using as queries the BLAST algorithm(http://www.ncbi.nlm.nih.gov/blast/blast.cgi) which revealed no sequencewith significant homology. The results of homology analyses indicatethat the sequences of

WDRPUH-A24-9-40, (SEQ ID NO: 1) WDRPUH-A24-9-314, (SEQ ID NO: 2)WDRPUH-A24-9-509, (SEQ ID NO: 3) WDRPUH-A24-9-339, (SEQ ID NO: 4)WDRPUH-A24-10-409, (SEQ ID NO: 16) WDRPUH-A24-10-40, (SEQ ID NO: 17)WDRPUH-A02-9-39, (SEQ ID NO: 30) WDRPUH-A02-9-407, (SEQ ID NO: 31)WDRPUH-A02-9-288, (SEQ ID NO: 34) WDRPUH-A02-9-237, (SEQ ID NO: 36)WDRPUH-A02-9-543, (SEQ ID NO: 37) WDRPUH-A02-10-570, (SEQ ID NO: 40)WDRPUH-A02-10-263, (SEQ ID NO: 41) WDRPUH-A02-10-78, (SEQ ID NO: 45)WDRPUH-A02-10-10, (SEQ ID NO: 49) WDRPUH-A02-10-411, (SEQ ID NO: 55)WDRPUH-A02-10-287  (SEQ ID NO: 57) and WDRPUH-A02-10-265 (SEQ ID NO: 61)are unique and thus, there is little possibility, to our best knowledge,that these molecules raise unintended immunologic responses to someunrelated molecules.

In conclusion, novel HLA-A24 and A2 epitope peptides were identified anddemonstrated to be applicable for cancer immunotherapy.

INDUSTRIAL APPLICABILITY

The present invention describes new TAAs, particularly those derivedfrom WDRPUH which induce potent and specific anti-tumor immune responsesand have applicability to cancer types such as hepatocellular carcinoma.Such TAAs warrants further development of the clinical application ofpeptide vaccination strategy in cancer.

While the invention has been described in detail and with reference tospecific embodiments thereof, it is to be understood that the foregoingdescription is exemplary and explanatory in nature and is intended toillustrate the invention and its preferred embodiments. Through routineexperimentation, one skilled in the art will readily recognize thatvarious changes and modifications can be made therein without departingfrom the spirit and scope of the invention. Thus, the invention isintended to be defined not by the above description, but by thefollowing claims and their equivalents.

What is claimed is:
 1. A method of inducing an immune response against ahepatocellular cancer in a subject, said hepatocellular cancerexpressing WD repeat protein upregulated in hepatocellular carcinoma(WDRPUH) comprising the amino acid sequence set forth in SEQ ID NO: 64,said method comprising the step of administering to the subject acomposition comprising an isolated peptide of less than 15 amino acidsselected from the group consisting of: (a) an isolated peptide, whichcomprises the amino acid sequence as shown in SEQ ID NO: 2; and (b) anisolated peptide, which comprises the amino acid sequence as shown inSEQ ID NO: 2 in which 1 or 2 amino acid(s) are substituted and/or added,wherein the added amino acid(s) are added to the N-terminus and/or theC-terminus.
 2. The method of claim 1, wherein said composition comprisesan adjuvant.
 3. The method of claim 1, wherein said composition isadministered by subcutaneous injection.
 4. The method of claim 1,wherein the dosage of said peptide is 0.1 mg to 10 mg.
 5. The method ofclaim 1, wherein the isolated peptide consists of the amino acidsequence as shown in SEQ. ID NO: 2.